A nucleic acid interference pharmaceutical composition for inhibiting PCSK9 gene expression and its application

Abstract

The present invention discloses a nucleic acid interference pharmaceutical composition for inhibiting PCSK9 gene expression and its application. To address the problems of existing statins and antibody drugs used to inhibit PCSK9 expression, such as insufficient duration of efficacy, poor patient compliance, high cost, and unstable efficacy, the present invention has developed a nucleic acid interference pharmaceutical composition for inhibiting PCSK9 gene expression that has low toxicity, good in vivo efficacy, and long-lasting in vivo efficacy. The composition comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is a nucleic acid molecule capable of inhibiting and silencing PCSK9 gene expression, and the carrier is a polypeptide polymer. The nucleic acid interference pharmaceutical composition of the present invention has high drug safety and high bioavailability, and as a new drug, has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a nucleic acid interference pharmaceutical composition for inhibiting PCSK9 gene expression and its application. Background Art

[0002] Proprotein convertase subtilisin / kexin type 9 (PCSK9) is the ninth member of the mammalian serine protease family. Serine proteases are a group of protein convertases (PCs) that cleave inactive secretory precursors into bioactive proteins and peptides (Said et al., 2003, Proc Natl Acad Sci USA; 100:928–933). PCSK9 was first discovered in primary cerebellar neurons in 2003. Its mRNA expression is upregulated during apoptosis (Jiang et al., 2001; Sayida et al., 2003), leading to its initial designation as neuronal apoptosis regulator convertase-1 (NARC-1). PCSK9 primarily interacts with the low-density lipoprotein receptor (LDLR) (Kosenko et al., 2013; Ferrier et al., 2016; Burnip et al., 2020), acting as a negative regulator of the LDLR. It has been shown to play an important role in cholesterol metabolism, cholesterol biosynthesis enzymes, and the low-density lipoprotein receptor (LDLR). In vivo experimental mouse models, PCSK9 mRNA expression levels can be knocked down (Maxwell KN, 2003, J. Adipose Res., 44: 2109-2119) and upregulated (Holden JD, 2003, PNAS USA; 100: 12027-12032). Some overexpression studies have shown that PCSK9 can control LDLR levels, thereby controlling the uptake of LDL by the liver (Maxwell KN, 2004, PNAS USA; 101: 7100-7105; Benjamin S et al., 2004, J. Biol. Chem., 279: 48865-48875; Parker SW, 2004, J. Biol. Chem., 279: 50630-50638). In addition, researchers (Yang Wei et al., 2021, Protein & Cell) analyzed the TCGA database and clinical tumor samples and found that PCSK9 was highly expressed in a variety of tumor tissues and was negatively correlated with the degree of infiltration of immune cells in the tumor microenvironment. Further mechanism studies found that LDLR in CD8 + T cells also play a non-classical regulatory role in addition to transporting LDL, that is, in addition to CD8 + In addition to the lipoprotein transport function of LDLR, T cell proliferation also depends on the expression of LDLR, and its effector functions including cytokine expression and target cell killing also mainly depend on the expression of LDLR. Therefore, the high expression of PCSK9 in the tumor microenvironment will lead to CD8 + The number of LDLR on the surface of T cells decreases, thereby inhibiting CD8 +PCSK9 promotes T cell proliferation, activation, and target cell cytotoxicity, leading to immune evasion of tumor cells. The most notable effect of PCSK9 is its interaction with the low-density lipoprotein receptor (LDLR) in the liver (Abou-Fadl et al., 2003, Nature Genetics; 34:154-156). Hepatocytes secrete PCSK9 into the circulation, where it binds to the LDLR to form the PCSK9 / LDLR complex. The catalytic domain of PCSK9 interacts with the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR. When the PCSK9 / LDLR complex is internalized, the low pH of the endosome increases its affinity, and PCSK9 prevents the open, extended conformation of the LDLR involved in receptor retrieval. Consequently, the PCSK9 / LDLR complex is transported to the lysosome for degradation, resulting in a decrease in surface LDLR and an increase in plasma cholesterol levels (Sayida et al., 2003; Benjamin et al., 2004; Poirier et al., 2006; Losurdo et al., 2011). This is because PCSK9 primarily affects LDL-C clearance efficiency by binding to the LDLR, and this interaction induces LDLR degradation, resulting in a closely interrelated regulation of PCSK9, LDLR, and LDL-C levels (Taveri et al., 2013). Atherosclerotic cardiovascular disease (ASCVD) is one of the diseases with the highest morbidity and mortality rates worldwide. The occurrence and progression of ASCVD are closely related to dyslipidemia. The mainstream view of dyslipidemia is the LDL-C (low-density lipoprotein cholesterol) theory, which holds that LDL-C and ASCVD have a causal relationship. A meta-analysis showed that for every 1 mmol / L (38 mg / dL) reduction in LDL-C, the incidence of coronary heart disease (CHD) events decreased by approximately 22%.

[0003] Statins lower LDL-C levels by inhibiting hydroxymethylglutaryl coenzyme (AMG-CoA) reductase. Since their introduction in 1987, statins have been a core lipid-lowering medication. Statins are the primary and secondary first-line treatments for ASCVD, but some patients are intolerant to statins or whose maximum tolerated doses do not reach the LDL-C therapeutic target (<1.8 mmol / L (70 mg / dL) or <1.4 mmol / L (55 mg / dL)).

[0004] Elevated cholesterol levels, particularly low-density lipoprotein (LDL) levels (LDL > 4.1 mmol / L or 160 mg / dL), are directly associated with an increased risk of cardiovascular disease. Statins are inadequately controlling severe hypercholesterolemia caused by mutations in the hepatic low-density lipoprotein receptor (LDLR), which impair LDLR-mediated clearance of LDL particles from the blood (Goldstein JL et al., J. Biol. Chem.; 1974, 249: 5153). This inherited condition, known as familial hypercholesterolemia (FH), can also be caused by mutations in apolipoprotein B, the major protein component of LDL particles that facilitates their binding to the LDLR. When PCSK9 binds to the LDL-loaded LDLR during endocytosis, the complex is directed to the lysosome for degradation. However, when the LDL-loaded LDLR is unbound by PCSK9, the LDLR unloads the LDL particle and returns it to the cell surface. Therefore, gain-of-function PCSK9 mutations lead to increased LDLR degradation, thereby impairing the uptake of LDL particles from the blood. The accumulation of low-density lipoprotein cholesterol in the bloodstream can accelerate the progression of atherosclerosis and lead to rupture of atherosclerotic lesions, triggering cardiovascular events and thus premature death.

[0005] In 2003, Abifadel et al. discovered that PCSK9 gene mutations are associated with autosomal dominant hypercholesterolemia (ADH). Major discoveries over the past decade have revealed the following: a. Gain-of-function mutations in PCSK9 are a cause of ADH; b. Loss-of-function mutations in PCSK9 are associated with reduced low-density lipoprotein cholesterol (LDL-C) levels and significantly lower cardiovascular risk. Loss-of-function mutations in PCSK9 have been studied in mouse models (Rashid et al., 2005, Proc. Natl. Acad. Sci. USA; 102: 5374-5379) and have been identified in humans (Cohen et al., 2005, Nat. Genet.; 37: 161-165). In both cases, loss of PCSK9 function leads to lower levels of total cholesterol and LDL-C. A retrospective study of data from the past few decades showed that sense mutations in PCSK9 can lower LDL-C levels and increase risk-benefit protection against the development of cardiovascular heart disease (Cohen et al., 2006, New England Journal of Medicine; 354: 1264-1272).

[0006] After the discovery of the positive effects of loss-of-function mutations in PCSK9, two fully human monoclonal antibodies targeting PCSK9 were developed: alirocumab (Praluent), developed in collaboration between Regeneron and Sanofi and approved by the FDA in July 2015 and by the National Medical Products Administration (NMPA) in December 2019, and evolocumab (Repatha), developed by Amgen and approved by the FDA in August 2015 and by the National Medical Products Administration (NMPA) in July 2018. Multiple clinical trials have demonstrated that PCSK9 inhibitors can lower LDL-C levels and have a synergistic effect with statins.

[0007] However, PCSK9 inhibition fails to lower LDL-C levels in patients with LDLR-negative homozygous FH. Furthermore, PCSK9 monoclonal antibodies require frequent dosing (subcutaneous injection every two weeks) and a high annual cost (approximately $5,700), making them more expensive than statins and leading to poor patient compliance. Consequently, both monoclonal antibodies have experienced slow sales growth and underperforming in the market.

[0008] In the field of lipid-lowering drugs, although antibodies and statins targeting PCSK-9 already exist, patient compliance is a key issue in the treatment of such chronic diseases. Small nucleic acid drugs have unique advantages in this regard, and have good efficacy and safety for patients with cardiovascular disease and those intolerant to statins. More importantly, small nucleic acid drugs have a long-term effect of up to several months for targeting PCSK9, which is unmatched by antibodies and statins.

[0009] With a deeper understanding of the PCSK9 target and the fierce competition among PCSK9 antibody drugs, scientists are developing more PCSK9-targeted therapies. Small nucleic acid drugs have reached a transformative milestone. Inclisiran, a highly anticipated drug, is a collaboration between leading small nucleic acid company Alnylam and The Medicines Company. The Medicines Company initiated the ORION-1 Phase II trial in 2016. In November 2019, Novartis acquired The Medicines Company for $9.7 billion, acquiring inclisiran. Based on the results of three Phase III clinical trials (ORION-9, ORION-10, and ORION-11), inclisiran received marketing approval from the European Union in December 2020 and the FDA a year later. The first dose was administered in China in July 2021, and a Phase III clinical trial (ORION-18) is currently underway in China. Inclisran has comparable efficacy to PCSK9 antibodies, but importantly, it only requires dosing every six months, significantly improving patient compliance. Since there are currently few small nucleic acid drugs targeting PCSK9, which limits users' choices and also leads to high costs for existing small nucleic acid drugs, the promotion of PCSK9 targeted therapy is slow. Therefore, it is necessary to develop more small nucleic acid drugs targeting PCSK9 targets, enrich the types of small nucleic acid drugs, provide users with more choices, and contribute to the development of nucleic acid drugs. Summary of the Invention

[0010] The purpose of the present invention is to provide a nucleic acid interference pharmaceutical composition for inhibiting PCSK9 gene expression with low toxicity, good in vivo efficacy and long-lasting in vivo efficacy, enrich the types of small nucleic acid drugs, and provide users with more choices.

[0011] Another object of the present invention is to provide the application of the above-mentioned nucleic acid interference pharmaceutical composition for inhibiting PCSK9 gene expression.

[0012] Another object of the present invention is to provide a drug for treating or preventing PCSK9-mediated cardiovascular diseases.

[0013] In order to achieve the above object, the technical solution adopted by the present invention is:

[0014] A nucleic acid interference pharmaceutical composition for inhibiting PCSK9 gene expression comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is a nucleic acid molecule capable of inhibiting and silencing PCSK9 gene expression, and the carrier is a polypeptide polymer.

[0015] Preferably, the polypeptide polymer is a histidine-lysine polymer, more preferably an H3K4b-type histidine-lysine polymer.

[0016] According to some embodiments, the carrier is HKP and / or HKP(+H).

[0017] Preferably, the nucleic acid molecule comprises one or more of siRNA, antisense oligonucleotide, miRNA, nucleic acid aptamer or decoy oligonucleotide. Preferably, the siRNA is designed based on the DNA sequence of the target gene PCSK9.

[0018] More preferably, the siRNA molecule is an oligonucleotide having a chain length of 17 to 28 base pairs, for example, the chain length of the siRNA molecule is 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 base pairs.

[0019] Further preferably, the siRNA molecule comprises a sense strand and an antisense strand.

[0020] Specifically, the sequence of the sense strand is selected from any one of SEQ ID No. 1 to 97 shown in Table 1:

[0021] Table 1

[0022]

[0023]

[0024]

[0025] Specifically, the antisense strand is selected from SEQ ID No. 98 to 198 shown in Table 2 that is complementary to the sense strand:

[0026] Table 2

[0027]

[0028]

[0029]

[0030] Still further preferably, the sense strand and / or antisense strand of the siRNA molecule is chemically modified.

[0031] According to some embodiments, the chemical modification includes base 2'-OME modification and / or base 2'-F modification.

[0032] Furthermore, the ratio of the number of bases with 2'-F modification to the number of bases with 2'-OME modification in the sense strand of the siRNA molecule is 1:4-7.

[0033] Furthermore, one or more bases in the 5th to 12th bases in the sense strand (5'→3') of the siRNA molecule are modified with 2'-F, and the remaining bases in the sense strand are modified with 2'-OME.

[0034] Furthermore, at least three consecutive bases in the sense strand of the siRNA molecule are modified with 2'-F.

[0035] Furthermore, the ratio of the number of bases with 2'-F modification to the number of bases with 2'-OME modification in the antisense strand of the siRNA molecule is 1:2-12.

[0036] Furthermore, at least two bases in the antisense strand (5'→3') of the siRNA molecule are modified with 2'-F.

[0037] Furthermore, the 2'-F modified bases in the antisense strand of the siRNA molecule are dispersed at different sites, and no more than two consecutive sites are subjected to 2'-F modification.

[0038] According to some embodiments, the siRNA is blunt-ended.

[0039] According to other embodiments, the siRNA has sticky ends, preferably with two unpaired bases hanging at the end of the antisense strand.

[0040] According to some embodiments, each base of the sense strand and / or antisense strand of the siRNA molecule is chemically modified.

[0041] According to some preferred embodiments, a 19-base-pair siRNA with blunt ends is selected, wherein bases 7 to 9 in the sense strand (5'→3') are modified with 2'-F, and the remaining bases are modified with 2'-OME, and bases 2 and 14 in the antisense strand (5'→3') are modified with 2'-F, and the remaining bases are modified with 2'-OME.

[0042] According to some preferred embodiments, the sense strand of the siRNA is 19 bases, and the antisense strand is 21 bases. The bases 7 to 9 in the sense strand (5'→3') are modified with 2'-F, and the remaining bases are modified with 2'-OME. The bases 2 and 14 in the antisense strand (5'→3') are modified with 2'-F, and the remaining bases are modified with 2'-OME.

[0043] According to other preferred embodiments, the sense strand of the siRNA is 19 bases, and the antisense strand is 21 bases. The bases at positions 5 and 7 to 9 in the sense strand (5'→3') are modified with 2'-F, and the remaining bases are modified with 2'-OME. The bases at positions 2, 6, 8, 9, 14, and 16 in the antisense strand (5'→3') are modified with 2'-F, and the remaining bases are modified with 2'-OME.

[0044] According to some preferred embodiments, the sense strand of the siRNA is 21 bases, and the antisense strand is 23 bases. The bases at positions 9 to 11 (5'→3') in the sense strand are modified with 2'-F, and the remaining bases are modified with 2'-OME. The bases at positions 2 and 14 (5'→3') in the antisense strand are modified with 2'-F, and the remaining bases are modified with 2'-OME.

[0045] According to other preferred embodiments, the sense strand of the siRNA is 21 bases, and the antisense strand is 23 bases. The bases at positions 7 and 9 to 11 in the sense strand (5'→3') are modified with 2'-F, and the remaining bases are modified with 2'-OME. The bases at positions 2, 6, 14, and 16 in the antisense strand (5'→3') are modified with 2'-F, and the remaining bases are modified with 2'-OME.

[0046] According to some specific and preferred embodiments, the siRNA molecules are one or more of the following oligonucleotides:

[0047] Justice chain: GmCmCmUmGmGmAfGfUfUmUmAmUmUmCmGmGmAmAm,

[0048] Antisense strand: UmUfCmCmGmAmAmUmAmAmAmCmUmCfCmAmGmGmCm;

[0049] Justice Chain: CmCmCmUmCmAmUfAfGfGmCmCmUmGmGmAmGmUmUm,

[0050] Antisense strand: AmAfCmUmCmCmAmGmGmCmCmUmAmUfGmAmGmGmGmUmGm;

[0051] Justice chain: GmCmAmCmCmCmUfCfAfUmAmGmGmCmCmUmGmGmAm,

[0052] Antisense strand: UmCfCmAmGmGmCmCmUmAmUmGmAmGfGmGmUmGmCmCmGm;

[0053] Justice chain: CmAmCmCmCmUmCmAmUfAfGfGmCmCmUmGmGmAmGmUmUm,

[0054] Antisense strand: AmAfCmUmCmCmAmGmGmCmCmUmAmUfGmAmGmGmGmUmGmCmCm;

[0055] Justice chain: CmGmGmCmAmCmCmCmUfCfAfUmAmGmGmCmCmUmGmGmAm,

[0056] Antisense strand: UmCfCmAmGmGmCmCmUmAmUmGmAmGfGmGmUmGmCmCmGmCmUm;

[0057] Justice chain: CmCmCmUmCfAmUfAfGfGmCmCmUmGmGmAmGmUmUm,

[0058] Antisense strand: AmAfCmUmCmCfAmGfGfCmCmUmAmUfGmAfGmGmGmUmGm;

[0059] Justice chain: GmCmAmCmCfCmUfCfAfUmAmGmGmCmCmUmGmGmAm,

[0060] Antisense strand: UmCfCmAmGmGfCmCfUfAmUmGmAmGfGmGfUmGmCmCmGm;

[0061] Justice chain: CmAmCmCmCmUmCfAmUfAfGfGmCmCmUmGmGmAmGmUmUm,

[0062] Antisense strand: AmAfCmUmCmCfAmGmGmCmCmUmAmUfGmAfGmGmGmUmGmCmCm;

[0063] Justice chain: CmGmGmCmAmCmCfCmUfCfAfUmAmGmGmCmCmUmGmGmAm,

[0064] Antisense strand: UmCfCmAmGmGfCmCmUmAmUmGmAmGfGmGfUmGmCmCmGmCmUm;

[0065] Justice chain:AmCmCmCmUmCmAfUfAfGmGmCmCmUmGmGmAmGmUm,

[0066] Antisense strand: AmCfUmCmCmAmGmGmCmCmUmAmUmGfAmGmGmGmUmGmCm;

[0067] Justice chain: GmCmAmCmCmCmUmCmAfUfAfGmGmCmCmUmGmGmAmGmUm,

[0068] Antisense strand: AmCfUmCmCmAmGmGmCmCmUmAmUmGfAmGmGmGmUmGmCmCmGm;

[0069] Justice chain:AmCmCmCmUfCmAfUfAfGmGmCmCmUmGmGmAmGmUm,

[0070] Antisense strand: AmCfUmCmCmAfGmGfCfCmUmAmUmGfAmGfGmGmUmGmCm;

[0071] Justice chain: GmCmAmCmCmCmUfCmAfUfAfGmGmCmCmUmGmGmAmGmUm,

[0072] Antisense strand: AmCfUmCmCmAfGmGmCmCmUmAmUmGfAmGfGmGmUmGmCmCmGm,

[0073] Justice chain: GmCmCmUmGmGmAfGfUfUmUmAmUmUmCmGmGmAmAm;

[0074] Antisense strand: UmUfCmCmGmAmAmUmAmAmAmCmUmCfCmAmGmGmCmCmUm,

[0075] Justice chain:AmGmGmCmCmUmGmGmAfGfUfUmUmAmUmUmCmGmGmAmAm;

[0076] Antisense strand: UmUfCmCmGmAmAmUmAmAmAmCmUmCfCmAmGmGmCmCmUmAmUm;

[0077] Justice chain: GmCmCmUmGmGmAfGfUfUmUmAmUmUmCmGmGmAmAmdTdT;

[0078] Antisense strand: UmUfCmCmGmAmAmUmAmAmAmCmUmCfCmAmGmGmCmdTdT,

[0079] Here, m represents a 2'-OME modification of the base, and f represents a 2'-F modification of the base.

[0080] Preferably, the carrier is HKP or HKP(+H).

[0081] The present invention also provides a use of the nucleic acid interference pharmaceutical composition in the preparation of drugs for treating and preventing cardiovascular diseases, blood lipid lowering drugs, cholesterol lowering drugs or anti-tumor drugs.

[0082] The present invention also provides a medicine, which includes the nucleic acid interference pharmaceutical composition, and is used to treat and prevent cardiovascular diseases, hyperlipidemia, hypercholesterolemia or tumor diseases.

[0083] The drug can effectively reduce the low-density lipoprotein cholesterol content and the total cholesterol content, has therapeutic effects on hypercholesterolemia, and further has therapeutic effects on cardiovascular diseases and hyperlipidemia caused by high cholesterol.

[0084] The drug can knock down the expression level of PCSK9 gene in tumor cells and has application prospects in preventing or treating tumor diseases, wherein the tumor diseases include but are not limited to cervical cancer, liver cancer and bladder cancer.

[0085] Preferably, the N / P ratio of the active ingredient and the carrier is 2 / 1 to 6 / 1, for example, 2 / 1, 2.5 / 1, 3 / 1, 3.5 / 1, 4 / 1, 4.5 / 1, 5 / 1, 5.5 / 1 or 6 / 1.

[0086] Preferably, the drug is a nanoparticle.

[0087] Further preferably, the size of the nanoparticles is 50 to 300 nm, for example, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205 nm, 210 nm, 215 nm, 220 nm, 225 nm, 230 nm, 235 nm, 240nm, 245nm, 250nm, 255nm, 260nm, 265nm, 270nm, 275nm, 280nm, 285nm, 290nm, 295nm, 300nm.

[0088] Further preferably, the dosage form of the drug is a lyophilized preparation.

[0089] Preferably, the drug is administered by subcutaneous injection and / or intravenous injection.

[0090] More preferably, the drug is administered by subcutaneous injection.

[0091] Preferably, the subject of administration of the drug is a mammal.

[0092] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0093] The present invention designs a series of siRNAs targeting the PCSK9 gene and uses histidine-lysine polypeptide (HKP(+H)) for in vivo delivery. These siRNAs have the advantages of low toxicity, good in vivo delivery efficacy, and the ability to effectively inhibit and silence PCSK9 gene expression in vivo. They can be used to treat or prevent PCSK9-mediated cardiovascular diseases, hyperlipidemia, hypercholesterolemia, and tumors. Furthermore, a unique siRNA modification is used to improve the stability of the siRNA in vivo while ensuring its knockdown effect on the PCSK9 gene, making the siRNA less susceptible to enzymatic degradation, thereby allowing it to continue to function in vivo.

[0094] The nucleic acid interference pharmaceutical composition of the present invention improves drug safety, reduces the "off-target" effect caused by the non-specific action of siRNA, and at the same time avoids the immune response triggered by exogenous RNA, and improves the bioavailability of siRNA, enabling it to effectively act on the diseased area, providing users with more options, reducing drug costs, and having broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 The figure shows the distribution of 92 siRNAs on the PCSK9 gene;

[0096] Figure 2 This is the EC50 screening result of siRNA sequence for PCSK9 at the cell level (Hela);

[0097] Figure 3 The figure shows the comparison results of EC50 curves of PCSK9 siRNA (PCSK9-25-hm13#) with different modification modes in Hela cell line;

[0098] Figure 4 The figure shows the comparison results of EC50 curves of PCSK9 siRNA (PCSK9-25-hm14#) with different modification modes in Hela cell line;

[0099] Figure 5 The figure shows the comparison results of EC50 curves of PCSK9 siRNA (PCSK9-19-hm13#) with different modification modes in Hela cell line;

[0100] Figure 6Schematic diagram of the in vivo pharmacodynamics experimental design for testing STP137 (NPs formed using the PNP HKP(+H) system and the candidate sequence 19-hm13#mod) in a hypercholesterolemia mouse model.

[0101] Figure 7 To verify the in vivo pharmacodynamics of STP137 in mice, blood was collected before administration (D0), on the 7th day (D7), 14th day (D14), and 28th day (D28) after the first administration, and every 2 weeks thereafter until the 140th day (D140).

[0102] Figure 8 Figure 1 shows the results of body weight change monitoring for in vivo pharmacodynamic validation of STP137 in mice.

[0103] Figure 9 Figure 1 shows the results of the in vivo pharmacodynamics validation of STP137 in mice, showing the plasma HDL-C, TC, and TG levels at the 145th day (D145) after the first dose.

[0104] Figure 10 To validate the in vivo pharmacodynamics of STP137 in mice, the inhibitory effect of target gene PCSK9 mRNA in the liver was shown on day 145 (D145) after the first dose.

[0105] Figure 11 This is a pathological staining image of liver tissue sections;

[0106] Figure 12 Schematic diagram of the in vivo pharmacodynamics experimental design for STP137-1, STP137-2, and STP137-25m1 in a hypercholesterolemia mouse model;

[0107] Figure 13 To validate the in vivo pharmacodynamics of STP137-1, STP137-2, and STP137-25m1 in mice, the relative changes in serum LDL-C levels before administration (D0), 7 days (D7), 14 days (D14), and 42 days (D42) after the first administration are shown.

[0108] Figure 14 To verify the in vivo pharmacodynamics of STP137-1, STP137-2, and STP137-25m1 in mice, the relative expression levels of PCSK9 in serum before administration (D0), 7 days (D7), and 14 days (D14) after the first administration were shown. DETAILED DESCRIPTION

[0109] The present invention is further described below with reference to the following examples. However, the present invention is not limited to the following examples. The implementation conditions used in the examples may be further adjusted according to the specific requirements of the application. Unspecified implementation conditions are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention may be combined with each other as long as they do not conflict with each other.

[0110] Existing statins and antibody drugs used to inhibit PCSK9 expression suffer from issues such as limited duration of efficacy, poor patient compliance, high costs, unstable efficacy, and a limited selection of commercially available siRNA drug products. After extensive research and experimental verification, the inventors have developed a nucleic acid interference pharmaceutical composition for inhibiting PCSK9 gene expression that exhibits low toxicity, excellent in vivo efficacy, and prolonged duration of efficacy.

[0111] According to the embodiments, the nucleic acid interference pharmaceutical composition for inhibiting PCSK9 gene expression of the present invention includes an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is a nucleic acid molecule capable of inhibiting and silencing PCSK9 gene expression, and the carrier is an H3K4b-type histidine-lysine polymer.

[0112] The carrier used in the present invention is a brand-new nanomaterial, a cationic polymer that promotes the absorption of small nucleic acid drugs. It can form nanoparticles with negatively charged small nucleic acid molecules through electrostatic interaction. To date, a large amount of in vitro research data has shown that this nanocarrier has the characteristics of efficient introduction, safety and non-toxicity. The innovative application of HKP or HKP (+H) has been unanimously recognized by the industry and represents a new type of small nucleic acid nano-delivery system. However, no drug has yet been used in human trials using HKP or HKP (+H) as a raw material or part of a formulation. At present, it is not possible to guarantee the efficient delivery of nucleic acid drugs in vivo and the stable, efficient and sustained efficacy of the drugs in vivo.

[0113] The present invention adopts a specific algorithm and programming containing several parameter conditions to design a series of siRNA sequences for the target gene PCSK9. The siRNA molecules targeting the PCSK9 gene are oligonucleotide sequences with a length of 19 to 25 base pairs.

[0114] The present invention also adopts unique siRNA modification to improve the stability of siRNA molecules, making siRNA less susceptible to enzyme degradation and able to continue to play a role in the body.

[0115] The delivery vector of the present invention is HKP or HKP(+H).

[0116] Specifically, through PNP technology, siRNA molecules targeting the PCSK9 gene are encapsulated into HKP or HKP+H (histidine-lysine polymer) carriers to prepare nanoparticle preparations.

[0117] Wherein, the N / P ratio of the active ingredient and the carrier is 2 / 1 to 6 / 1.

[0118] The present invention also provides the use of the nucleic acid interference pharmaceutical composition in the preparation of drugs for treating and preventing cardiovascular diseases, blood lipid lowering drugs, cholesterol lowering drugs or anti-tumor drugs.

[0119] Especially compared with traditional statins and antibody drugs, the nucleic acid interference drug composition of the present invention has many advantages such as good efficacy, low toxicity, and long duration of efficacy (up to 140 days). As a new drug, it has strong competitiveness.

[0120] The technical solutions and effects of the present invention are described in detail below through examples.

[0121] Example 1

[0122] Design siRNA sequences for target PCSK9

[0123] We used a specific algorithm and programming containing several parameter conditions to design a series of siRNA sequences for the target gene PCSK9, including oligonucleotide sequences of 25 base pairs, 21 base pairs, and 19 base pairs in length. These siRNA sequences targeting the PCSK9 gene can theoretically bind to and degrade PCSK9 mRNA in target cells through the RNAi mechanism, thereby blocking the translation level of the protein and inhibiting the protein expression of PCSK9.

[0124] 92 siRNAs were screened based on gene coding sequence, reasonable thermodynamic stability and low expected toxic side effects. Table 3 lists the 92 siRNA sequences targeting PCSK9. Their distribution on the PCSK9 gene is shown in Figure 1 .

[0125] Table 3. Designed and screened siRNA sequences targeting PCSK9

[0126]

[0127]

[0128]

[0129] Example 2

[0130] In vitro screening of siRNA sequences targeting PCSK9 gene (cellular level)

[0131] In this example, a commercial cell transfection reagent Lipo2000 was used, and cervical cancer cells (Hela), liver cancer cells (Hep1-6, HepG2), and bladder cancer cells (KU7) were selected for in vitro screening of target siRNA sequences.

[0132] Cells were transfected with the siRNA sequences listed in Table 3 using a transfection reagent (Lipo2000). Cell-based screening was performed for the siRNA sequences listed in Table 3 using in vitro cellular assays (including QRT-PCR, cell proliferation activity, and protein expression assays such as Western blotting to measure target gene mRNA expression). EC50 (the concentration that achieves 50% effective target gene knockdown) was then used to identify candidate siRNA sequences suitable for in vivo pharmacodynamic studies.

[0133] First, HeLa cells were seeded in 24-well cell plates (1×10 5 Cells were transfected with the siRNAs (19mer, 21mer, and 25mer) listed in Table 3 or a negative control using a commercial transfection reagent (lipo2000) at a concentration of 100 nM. Untreated cells served as blank controls. Total RNA was extracted from each cell 24 hours after transfection and reverse transcribed using a kit according to the manufacturer's instructions to obtain cDNA. Real-time PCR was used to determine the relative expression level of the target gene PCSK9 mRNA in each transfected HeLa cell, normalized to the housekeeping gene β-actin. Gene knockdown efficacy is expressed as a percentage of the blank control. The results are shown in Table 4.

[0134] Table 4. Preliminary screening results of siRNA sequences targeting PCSK9 at the cell level (Hela)

[0135]

[0136]

[0137]

[0138] In Table 4, “0%” and negative values ​​indicate no knockdown effect, and “\” indicates that the experiment was not performed.

[0139] Sequences with a knockdown effect (KD effect in Hela (%)) of 75% or more were selected for subsequent EC50 data detection and analysis. The effect of a single sequence was confirmed by setting multiple concentration gradients, and the half-maximum effective biological activity concentration (EC50, nM) of each candidate sequence was obtained. The results are shown in the figure below. Figure 2 As shown in the figure, the EC50 values ​​of all 27 sequences detected in Hela cells were less than 10 nM, indicating that they all had good knockdown effects. However, considering the use in subsequent in vivo animal experiments, human-mouse homologous (hm) sequences were selected as candidate sequences. Therefore, PCSK9-19-hm13#, PCSK9-25-hm13#, and PCSK9-25-hm14# were selected for EC50 detection of different modification methods (see Table 5 for modified sequences).

[0140] Table 5. Different modification methods of the three sequences screened by EC50 detection

[0141]

[0142]

[0143] Note: 2'-F(f), 2'-OME(m).

[0144] Example 3

[0145] In vitro screening of siRNA modified sequences targeting PCSK9 gene (cellular level)

[0146] Hela cells were seeded into 24-well cell plates (1×10 5 siRNA candidates (modified or unmodified) were transfected into different cells at multiple concentration gradients ( ) using the same procedures as the primary screening. GraphPad Prism8 software was used to plot data curves and calculate EC50 values. The optimal modification method was screened by comparing the EC50 curves of different modification methods.

[0147] The EC50 curves of the unmodified sequence PCSK9-25-hm13# and its different modification methods are shown in Figure 2. Figure 3 As shown in the figure, the EC50 of PCSK9-25-hm13#-212m1, PCSK9-25-hm13#-212m2, and PCSK9-25-hm13#-211m2 are all smaller than that of the unmodified sequence PCSK9-25-hm13#, indicating that the knockdown effects of these three modified sequences are better than those of the unmodified sequence.

[0148] The EC50 curves of the unmodified sequence PCSK9-25-hm14# and its different modification methods are shown in Figure 2. Figure 4As shown, the EC50 of PCSK9-25-hm14#-211m1, PCSK9-25-hm14#-231m1, PCSK9-25-hm14#-211m2, and PCSK9-25-hm14#-231m2 were significantly greater than 10 nM, and the knockdown effect after modification was poor.

[0149] The EC50 curves of the unmodified sequence PCSK9-19-hm13# and its modified sequence PCSK9-19-hm13#m1, the unmodified sequence PCSK9-19-hm13#-21 and its modified sequence PCSK9-19-hm13#-21m1, the unmodified sequence PCSK9-19-hm13#-23 and its modified sequence PCSK9-19-hm13#-23m1, and the unmodified sequence PCSK9-21-hm13# and its modified sequence PCSK9-21-hm13#m1 are shown as follows: Figure 5 As shown, the EC50 of PCSK9-19-hm13#-21m1 is smaller than that of the unmodified sequence and significantly smaller than 10 nM, so PCSK9-19-hm13#-21m1 is preferably used for in vivo experiments; although the EC50 of PCSK9-19-hm13#-23m1 is smaller than that of the unmodified sequence, it is larger than 10 nM, indicating that the knockdown effect is poor, and it is not used as a candidate sequence for in vivo experiments; the EC50 of PCSK9-21-hm13#m1 is smaller than that of the unmodified sequence and smaller than 10 nM, so it can also be used as a candidate sequence for in vivo experiments; the EC50 of PCSK9-19-hm13#m1 is larger than that of the unmodified sequence, but still smaller than 10 nM, indicating that it still has an excellent knockdown effect, and the modified siRNA has improved stability, is not easily degraded by enzymes, and can continue to function in vivo, so PCSK9-19-hm13#m1 can be selected for in vivo experiments.

[0150] In summary, after screening, PCSK9-19-hm13#m1, PCSK9-19-hm13-21m1, PCSK9-25-hm13#-212m2, PCSK9-25-hm13#-212m1 and PCSK9-25-hm13#-211m2, PCSK9-21-hm13#m1 will be used as candidate sequences in in vivo experiments.

[0151] Example 4

[0152] Preparation and identification of nanoparticles

[0153] The candidate sequences were mixed with polypeptide carriers (HKP+H) at a ratio of N / P = 2.5:1 (polypeptide carrier / candidate sequence) to form stable nanoparticle preparations (see patent CN 112703196 A) for in vivo pharmacodynamic verification.

[0154] Example 5

[0155] In vivo pharmacodynamics experiments

[0156] According to Example 4, PCSK9-19-hm13#m1 and HKP(+H) were self-assembled into nanoparticles, and the prepared nanoparticles were labeled as STP137.

[0157] This experiment used a hypercholesterolemia model established by C57BL / 6 mice after 10 weeks of high-fat feeding. The experiment tested the LDL-C (Baseline) of the mice before administration, and the mice were evenly divided into groups according to LDL-C. Blood was collected on the 7th day (D7), 14th day (D14), 28th day (D28) after the first administration and every 2 weeks thereafter. The second administration was carried out 2 months after grouping (59th day). High-fat diet (HFD) was used throughout the experiment. In the experiment, the control group was administered with normal saline, and the administration method was subcutaneous injection (sc). STP137 was administered by subcutaneous injection and intravenous injection (iv). One experimental group was set up for each administration method, and the dosage was 3 mg / kg. The experimental design is detailed in the following. Figure 6 .

[0158] The blood samples collected at each time point were separated into plasma and the LDL-C content was measured using an LDL-C biochemical detection kit. The relative LDL-C content (the ratio of the LDL-C content in each drug-treated group to the LDL-C content in the vehicle group) was calculated using the vehicle group as the benchmark. The data were analyzed using the T test. The data results are shown in Figure 7 .

[0159] according to Figure 7 As shown, STP137-sc significantly lowered LDL-C 14 days after the first dose. LDL-C levels fluctuated thereafter, but data from the 14th day onward remained statistically significant compared to the vehicle group at every time point. Three months after the first dose, LDL-C levels were reduced to less than 50% compared to the control group and remained stable. The LDL-C-lowering effect of STP137-iv was less stable.

[0160] The body weight test results of the Vehicle group, STP137-sc group and STP137-iv group are shown in Figure 8 .from Figure 8 In terms of body weight, compared with the Vehicle group, the body weight of the STP137-sc group and the STP137-iv group were under certain control.

[0161] Endpoint blood samples were collected from each group on day 145 after the first dose, and plasma was separated for analysis of high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), and triglyceride (TG) levels. Liver PCSK9 mRNA was also measured on day 145 after the first dose.

[0162] The results of high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC) and triglyceride (TG) content are as follows: Figure 9 As shown in the endpoint data, after subcutaneous administration of STP137, the plasma total cholesterol (TC) level at the end point was significantly lower than that of the control group (Vehicle group). High-density lipoprotein cholesterol (HDL-C) did not change significantly compared to the control group (Vehicle group), and triglycerides (TG) decreased slightly, but not significantly. In contrast, after intravenous administration of STP137, the plasma high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), and triglyceride (TG) levels did not change significantly compared to the control group.

[0163] The results of PCSK9 mRNA expression in the liver are as follows Figure 10 As shown, subcutaneous administration of STP137 (145 days after the first dose and 86 days after the second dose) had an approximately 20% inhibitory effect on PCSK9 mRNA expression.

[0164] Liver tissue sections Figure 11 Compared with the Vehicle group, the degree of fatty liver in the STP137 subcutaneous administration group and the STP137 intravenous administration group was alleviated to varying degrees, among which the STP137 subcutaneous administration group was more significantly alleviated (the number of fat droplets was greatly reduced).

[0165] Example 6

[0166] In vivo pharmacodynamics experiment 2

[0167] According to Example 4, the nanoparticles formed by self-assembly of PCSK9-19-hm13#m1 and HKP(+H) are labeled as STP137-1, the nanoparticles formed by self-assembly of PCSK9-19-hm13-21m1 and HKP(+H) are labeled as STP137-2, and the nanoparticles formed by self-assembly of PCSK9-25-hm13#-212m1 and HKP(+H) are labeled as STP137-25m1.

[0168] This experiment used a hypercholesterolemia model established by feeding C57BL / 6 mice with a high-fat diet for 12 weeks. The LDL-C content of the mice was measured before administration (Day 0). The mice were evenly divided into groups based on LDL-C levels. Blood was collected on Day 7 (Day 7), Day 14 (Day 14), and Day 42 (D42) after the first administration. In the experiment, STP137-1, STP137-2, and STP137-25m1 were drug preparations produced by siRNA with three different candidate sequences. The NC group was a control group drug without a specific sequence and produced using the same vector and production process as STP137. The drug was administered by subcutaneous injection (sc) at a dose of 3 mg / kg. There were 10 mice in each group. The experimental design is detailed in [see ]. Figure 12 .

[0169] The blood samples collected at each time point were separated into plasma and the LDL-C content was detected using an LDL-C biochemical detection kit (Nanjing Jiancheng Bioengineering Institute). The relative LDL-C content (the ratio of the LDL-C content in each STP137 administration group to the LDL-C content in the NC group) was calculated using the NC group as the benchmark. The data were analyzed using the T test. The data results are shown in Figure 13 .

[0170] according to Figure 13 It can be seen that STP137-1 gradually reduced LDL-C after administration, and the reduction was significant at every time point compared with the NC group. On the 42nd day, LDL-C was reduced by about 50% compared with the NC group. STP137-2 lagged slightly behind STP137-1 in LDL-C reduction (no difference compared with the NC group on the 7th day), and on the 42nd day, it was also reduced by about 50% compared with the NC group. The degree of LDL-C reduction of STP137-25m1 was less. On the 14th and 42nd days, the LDL-C level was reduced by about 20% compared with the NC group, but there was no significant difference.

[0171] Serum PCSK9 data such as Figure 14 As shown in the data, the relative amounts of PCSK9 in STP137-1 and STP137-2 gradually decreased after administration. The PCSK9 content in STP137-1 decreased by about 55% on Day 14 relative to the NC group, and was significantly higher than that in the NC group. The PCSK9 content in STP137-2 decreased by about 48% on Day 14. The relative PCSK9 content in the STP137-25m1 group did not change on Day 7 and decreased by about 40% on Day 14.

[0172] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made in accordance with the spirit of the present invention are intended to be covered by the scope of protection of the present invention.

Claims

1. A nucleic acid interference pharmaceutical composition for inhibiting PCSK9 gene expression, comprising an active ingredient and a pharmaceutically acceptable carrier, characterized in that: The active ingredient is a siRNA molecule that can inhibit and silence PCSK9 gene expression, and the carrier is a polypeptide polymer. The siRNA molecule is PCSK9-19-hm13#m1, and its sequence is: Justice chain: GmCmCmUmGmGmAfGfUfUmUmAmUmUmCmGmGmAmAm, Antisense strand: UmUfCmCmGmAmAmUmAmAmAmCmUmCfCmAmGmGmCm, The polypeptide polymer is a histidine-lysine polymer.

2. The nucleic acid interference pharmaceutical composition according to claim 1, characterized in that The carrier is HKP and / or HKP(+H).

3. Use of the nucleic acid interference pharmaceutical composition according to claim 1 or 2 in the preparation of drugs for treating and preventing cardiovascular diseases, lipid-lowering drugs or cholesterol-lowering drugs.

4. A drug, characterized in that The medicine includes the nucleic acid interference pharmaceutical composition according to claim 1 or 2, and the medicine is used to treat and prevent cardiovascular disease, hyperlipidemia or hypercholesterolemia.

5. The drug according to claim 4, characterized in that The drug is a nanoparticle; and / or, the drug is administered by subcutaneous injection and / or intravenous injection; And / or, the subject of administration of the drug is a mammal.

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