WO2024067500A1 - Conjugué glycosyle d'acide nucléique antisens, son procédé de préparation et son utilisation dans le traitement du cancer du foie - Google Patents

Conjugué glycosyle d'acide nucléique antisens, son procédé de préparation et son utilisation dans le traitement du cancer du foie Download PDF

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WO2024067500A1
WO2024067500A1 PCT/CN2023/121152 CN2023121152W WO2024067500A1 WO 2024067500 A1 WO2024067500 A1 WO 2024067500A1 CN 2023121152 W CN2023121152 W CN 2023121152W WO 2024067500 A1 WO2024067500 A1 WO 2024067500A1
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nucleic acid
antisense nucleic
mmol
conjugate
compound
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Chinese (zh)
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杨振军
潘宇飞
管静
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北京大学
哈药(北京)生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical

Definitions

  • the present invention relates to a series of antisense nucleic acid glycosyl conjugates and preparation methods thereof, and also relates to a pharmaceutical preparation made from the antisense nucleic acid glycosyl conjugates and application thereof in treating liver cancer.
  • the present invention belongs to the field of biomedical technology.
  • Carbohydrate derivatives participate in many biological processes through lectins and carbohydrate-binding proteins, such as signal transduction and cell surface recognition [Chembiochem, 2004, 5(6): 740-764.].
  • the synthesis of carbohydrate derivative-oligonucleotide conjugates has been deeply explored.
  • Galactose has become the most studied carbohydrate derivative and is used to enhance the targeted delivery of oligonucleotides [J Am Chem Soc, 2012, 134(4): 1978-1981. Mol Ther, 2017, 25(1): 71-78.].
  • N-acetylgalactosamine is a targeting group with high affinity.
  • GalNAc conjugation can increase the efficacy of ON by ⁇ 7 times in mice and ⁇ 30 times in human patients [Nucleic Acids Res, 2014, 42(13): 8796-8807.]. Compared with nanoparticle complexes, GalNAc conjugates are smaller in size, have a clear and single chemical composition, and have low synthesis costs.
  • GalNAc conjugation strategy accounts for a large proportion of drug development in some pharmaceutical companies, such as Dicerna Pharmaceuticals, Silence Therapeutics, Arbutus Biopharma and Arrowhead Pharmaceuticals, which are all developing GalNAc conjugated products [Trends Pharmacol Sci, 2021, 42(7): 588-604.].
  • Alnylam which currently has three GalNAc conjugated drugs on the market, all of which are from the company, used to treat acute hepatic porphyria, hypercholesterolemia and hyperoxaluria [Nat Rev Drug Discov, 2020, 19(10): 673-694.].
  • the first GalNAc-conjugated siRNA Givosiran was approved by the FDA in 2019 for the treatment of AHP (acute hepatic porphyria) in adults.
  • AHP is a rare genetic disease caused by the accumulation of porphyrin metabolites in the patient's body due to defects in the representative enzymes in the heme production process [N Engl J Med, 2019, 380(6): 549-558.].
  • the principle of Givosiran is to induce silencing of the gene encoding aminolevulinate synthase 1, reduce ALA and The formation of porphobilinogen relieves the accumulation of porphyrin or its precursors.
  • Givosiran is 21/23 nucleotides in length and is chemically modified with PS, 2′-F and 2′-OMe. Phase III clinical results show that it can significantly reduce the occurrence of AHP and reduce the use of hemin (for the treatment of anemia) [Expert Opin Biol Ther, 2013, 13(3): 429-435.].
  • other pharmaceutical giants also have their own GalNAc-conjugated ON production pipelines and are in different stages of clinical research. The common feature they have is that they all appear in at least three clusters of continuous sugar molecule structures.
  • BRII-835 (VIR-2218) is a GalNAc-siRNA drug targeting HBV that is co-developed by Alnylam/Vir/Tengshengbo Pharmaceuticals and administered by subcutaneous injection. It mediates RNA interference and has the potential to directly antiviral activity against HBV and induce effective immune responses.
  • siRNA it is the first siRNA to enter the clinic using enhanced stable chemistry + (ESC +) technology, which can enhance stability and minimize off-target effects, thereby improving therapeutic efficacy.
  • ESC + enhanced stable chemistry +
  • a significant dose-dependent and sustained reduction in HBsAg was observed in HBeAg-negative and HBeAg-positive patients and at all doses within 24 weeks, and it was well tolerated by patients with chronic hepatitis B (EASL2021).
  • GalNAc conjugation does not seem to be a good choice for the treatment of liver cancer. Because the ASGPR receptor is expressed to varying degrees in various differentiated liver tumor cells, drug uptake may be seriously affected. However, there is also a view that the ASGPR receptor is extremely abundant on the surface of hepatocytes. Even if it is partially expressed at a low level, at normal dosing concentrations, the remaining amount of receptors is sufficient to mediate drug entry into the cell. The coupling of this ligand with small molecule drugs has also made good progress in the treatment of hepatocellular carcinoma [Mol Pharm, 2021, 18(1): 461-468.].
  • GalNAc conjugation did not show better gene silencing activity in vivo, which was attributed to the downregulation of ASGPR expression in highly differentiated human HCC tumor tissues [Cancer Res, 2022, 82(5): 900-915.].
  • the anti-liver cancer antisense nucleic acid CT102 developed by Wang Shengqi and others has completed all preclinical studies and entered Phase I clinical research in February 2021. It targets the mRNA of human insulin-like growth factor type I receptor (IGF1R), has clear efficacy and high safety, but the dosage concentration and frequency are high ( ⁇ 10 mg/kg for mice, every other day, a total of 10 tail vein injections). Studies have found that IGF1R and its ligands are abnormally expressed in malignant tumors such as acute leukemia, multiple myeloma, breast cancer, prostate cancer, ovarian cancer, endometrial cancer, cervical cancer, non-small cell lung cancer, and Ewing's sarcoma.
  • malignant tumors such as acute leukemia, multiple myeloma, breast cancer, prostate cancer, ovarian cancer, endometrial cancer, cervical cancer, non-small cell lung cancer, and Ewing's sarcoma.
  • IGF1R insulin-like growth factor
  • IGF1 or IGF2 insulin-like growth factor
  • PI3K P-phosphatidylinositol 3-kinase
  • AKT AKT
  • Rac mitogen-activated protein kinase
  • IGF1R interleukin-1 receptor
  • ER estrogen receptor
  • ERBB2 epidermal growth factor receptor 2
  • IGF1R is located upstream of the PI3K-AKT1-MTOR pathway, and the PI3K-AKT1-MTOR pathway is abnormally activated in more than half of breast cancer patients [Cancer Res, 2011, 71(21): 6773-6784]; preclinical data from sarcoma-tumor models showed that the IGF1R pathway is particularly important in tumor growth, metastasis, and angiogenesis in patients with Ewing sarcoma and rhabdomyosarcoma, and IGF1R inhibitors have been preliminarily used in these tumor patients [Lancet Oncol, 2010, 11(2): 129-135]; the expression level of IGF1R protein in non-small cell lung cancer (NSCLC) cell lines and patient samples is high in both adenocarcinoma and squamous tissues, and IGF1R expression is associated with poor prognosis in NSCLC patients [Thorac Cancer, 2020, 11(4): 875-887]. Despite this, the regulatory
  • One of the purposes of the present invention is to provide an antisense nucleic acid glycosyl conjugate and a method for preparing the same;
  • the second purpose of the present invention is to provide a combined delivery strategy of antisense nucleic acid combined with terminal conjugation
  • the third object of the present invention is to provide a pharmaceutical preparation prepared from the lipid complex containing the antisense nucleic acid drug and its use in the treatment of primary liver cancer.
  • the present invention discloses an antisense nucleic acid glycosyl conjugate, wherein the antisense nucleic acid glycosyl conjugate is formed by covalently coupling a sugar molecule to the 5′ end of an antisense nucleic acid via a connecting arm;
  • the sugar molecule is acetylgalactosamine (Gal), acetylglucosamine (Glu) or mannose (Man);
  • the structure of the connecting arm is as shown below L1, L2, L3, L4 or L5;
  • the left end of the connecting arm is connected to the sugar molecule, and the right end is connected to the 5' terminal hydroxyl group of the antisense nucleic acid.
  • the structure of the connecting arm is as shown in L1, L2, L3, L4 or L5
  • the structure of the connecting arm is as shown in L2, L4 or L5
  • the structure of the connecting arm is as shown in L2, L4 or L5
  • the structure of the connecting arm is as shown in L2, L4 or L5.
  • the antisense nucleic acid is selected from the following sequences:
  • CT102 TsCsCs TsCsCs GsGsAs GsCsCs AsGsAs CsTsTs CsA
  • CT102 MOE5 Tes m Ces m CesTes m Ces m C S G S G S A S G S m C S m C S A S G S A S m CesTesTes m CesAe;
  • m C represents 5-methylcytosine modification (5mC);
  • s represents phosphorothioate modification (PS);
  • e represents 2′-O-MOE modification (2′-O-MOE); the structures are as follows:
  • the antisense nucleic acid sugar conjugate is selected from the following compounds:
  • the method further comprises conjugating a fluorescent labeling molecule to the 3′ end of the antisense nucleic acid of the antisense nucleic acid sugar conjugate.
  • the structure of the antisense nucleic acid sugar conjugate conjugated with a fluorescent marker molecule is as follows:
  • the present invention also proposes the use of the antisense nucleic acid glycoconjugate in the preparation of a pharmaceutical preparation for treating tumors.
  • the tumor includes liver cancer.
  • the present invention also proposes a pharmaceutical preparation for anti-liver cancer, wherein the pharmaceutical preparation comprises a lipid complex formed by the antisense nucleic acid glycoconjugate of the present invention and DNCA, CLD and DSPE-PEG, wherein the structures of the DNCA, CLD and DSPE-PEG are as follows:
  • the ratio of the amount of DNCA, CLD, DSPE-PEG to the antisense nucleic acid substance in the antisense nucleic acid glycoconjugate is 30:30:0.6:1, 20:20:0.4:1, 40:20:0.6:1 or 20:40:0.6:1.
  • the present invention also proposes the use of the pharmaceutical preparation in preparing drugs for treating primary liver cancer.
  • the present invention constructs a series of 5′-terminal glycosyl conjugates based on the anti-liver cancer antisense nucleic acid sequence CT102 MOE5 with the best anti-liver cancer activity proved in previous experiments.
  • In vitro and in vivo experiments have shown that it has excellent serum stability and in vivo half-life. After a single intravenous administration, the drug can accumulate in the body for about 40 days.
  • sugar molecules increase the drug uptake capacity through glycosyl transporters on the cell surface. Through the screening of different linker arms and sugar molecules, some candidates with better activity were identified and in vivo efficacy experiments were carried out.
  • Each antisense nucleic acid conjugate can form stable spherical nanoparticles of about 150 nm under DNCA/CLD/DSPE-PEG encapsulation. This preparation showed good in vivo tumor inhibition effect under the intravenous administration regimen of 2mpk/4 days. Compared with the original unmodified chain CT102, it further reduced the frequency and dosage of administration and had good safety. Among them, Glu-CT102 MOE5 showed stable and optimal anti-liver cancer activity in the experiment and can be used as an anti-HCC antisense nucleic acid candidate for further study.
  • the present invention provides a series of new antisense nucleic acid sequences for CT102 target IGF1R mRNA, which target different regions of IGF1R mRNA exons respectively.
  • PHN02 and PHN07 are selected, which have more advantages than the original sequence CT102 in tumor cell proliferation inhibition, target gene silencing activity and apoptosis-promoting activity.
  • the in vivo and in vitro activity of PHN02 MOE5 obtained by Gapmer chemical modification is further improved, which is significantly better than the non-modified PHN02 and CT102.
  • the in vivo anti-tumor activity experiment shows that PHN02 MOE5 has a comparable tumor growth inhibition effect with the CT102 MOE5 selected in the early stage of the laboratory, and is worthy of further clinical research and development.
  • Fig. 1 is the synthetic route of G3Ac-NHS
  • FIG2 is a synthetic route of sGalNAc (sG) phosphoramidite monomer
  • FIG3 is a synthetic route of Gal(N/O), Glu(N/O), and Man(N/O) conjugated precursors
  • FIG4 is a synthetic route of Gal/Glu/Man conjugated precursor active ester
  • FIG5 is a flow chart of the synthesis of G3-ON by liquid phase solution convergence method
  • FIG6 is a flow chart of the synthesis of Cy5.5-G3-ON fluorescent marker by solution convergence method
  • FIG7 is an acrylamide gel electrophoresis to investigate the stability of CT102 modification and conjugate in 50% FBS;
  • the nucleic acid sample in each well is 10 pmol
  • FIG8 is a flow cytometric analysis of the uptake of various modifications and conjugate complexes of liposome-encapsulated CT102 in HepG2 and Huh7 cells after 4 hours;
  • A is the submicroscopic structure (100 nm scale) of the DNCA/CLD system encapsulating various modifications/conjugates of CT102 MOE5 ;
  • B is the submicroscopic structure (100 nm scale) of the DNCA/CLD system encapsulating various modifications/conjugates of CT102 MOE5 ;
  • FIG10 is the in vivo distribution and fluorescence quantitative analysis results of Cy5.5-labeled CT102 MOE5 and G3-CT102 MOE5 encapsulated by DNCA/CLD/DSPE-PEG (intravenous administration);
  • B. Curves of whole-body fluorescence quantitative changes over time in each group of mice. Data are expressed as mean ⁇ SD, n 3;
  • FIG11 is the results of in vitro tissue fluorescence quantitative analysis of Cy5.5-labeled G3-CT102 MOE5 and CT102 MOE5 encapsulated by DNCA/CLD/DSPE-PEG;
  • A Fluorescence imaging of the heart, lung, liver, spleen, kidney, and intestine of each group of mice at different time points (4h, 1d, 2d, 5d, 10d, 15d);
  • Figure 12 shows the inhibitory activity of each conjugate on HepG2 and Huh7 cell proliferation and target IGF1R mRNA silencing activity (100 nM). * represents P ⁇ 0.05;
  • FIG. 13 shows the in vivo antitumor effects of CT102 modifications and conjugates encapsulated by DNCA/CLD/PEG.
  • FIG14 shows the efficacy of the novel antisense nucleic acid sequence targeting IGF1R at the HepG2 and Huh-7 cell levels
  • FIG15 is a flow cytometry study of the apoptosis-promoting ability of the lipid material Mix encapsulated with CT102, PHN02, and PHN07 in HepG2;
  • Blank blank solvent control
  • NC Mix-encapsulated scrambled ASO
  • CT102, PHN02, and PHN07 are Mix-encapsulated antisense nucleic acid preparations CT102, PHN02, and PHN07, respectively.
  • FIG16 shows the effect of the novel antisense nucleic acid sequence targeting IGF1R on the proliferation activity and target gene silencing of A549, MCF-7 and B-CPAP cells;
  • A.Mix encapsulated antisense nucleic acid sequence had inhibitory activity on proliferation of A549, MCF-7 and B-CPAP cells;
  • B.Mix encapsulated antisense nucleic acid sequence had silencing effect on IGF1R mRNA in A549, MCF-7 and B-CPAP cells.
  • Figure 17 shows the efficacy of PHN02 MOE5 formulation at the HepG2 and Huh-7 cell levels
  • FIG18 shows the efficacy of Mix-encapsulated antisense nucleic acid PHN02 MOE5 on mice with orthotopic tissue transplanted liver cancer
  • A. Dosage regimen of anti-liver cancer antisense nucleic acid for treating orthotopic tissue transplanted liver cancer mice; B. Growth of the ratio of tumor fluorescence intensity at different time points after administration to the tumor fluorescence intensity before administration in each group; C. Ratio of tumor fluorescence intensity 28 days after administration to the tumor fluorescence intensity before administration; D. IGF1R mRNA expression in tumor tissues of mice in each group (n 4); E. Changes in body weight of mice during administration; ** represents P ⁇ 0.01, **** represents P ⁇ 0.0001;
  • FIG. 19 shows tumor imaging at different stages of the efficacy experiment of Mix-encapsulated antisense nucleic acid PHN02 MOE5 on mice with orthotopic tissue transplanted liver cancer.
  • the synthetic route of Gal/Glu/Man conjugation precursor active ester is shown in FIG4 .
  • Citric acid powder was added to the aqueous phase, the pH was adjusted to about 4, the product was extracted with DCM to the organic phase, and the product was dried by rotary evaporation. The product was obtained in an amount of 237 mg (0.53 mmol) with a yield of 75.7%.
  • Citric acid powder was added to the aqueous phase, the pH was adjusted to about 4, the product was extracted with DCM to the organic phase, and the product was dried by rotary evaporation. The product was obtained in an amount of 220 mg (0.49 mmol) with a yield of 70.3%.
  • the conjugated structure such as sG
  • the pure product is prepared, it is prepared into a 0.05g/mL solution with anhydrous acetonitrile.
  • the oligonucleotide synthesizer can be programmed to automatically conjugate to the 5′ end of the oligonucleotide (ON) using a general solid phase synthesis method. After the synthesis is completed, the standard process of nucleic acid chain is followed for separation and purification to obtain the sG-ON conjugate, and the total yield is about 20%. For compounds 17, 18, 20, 21, 23, and 24, an additional process for preparing phosphoramidite precursors is required.
  • the preparation process can be simply described as reacting with 1.5 equivalents of 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite and 1H-tetrazole in anhydrous acetonitrile under argon protection for 3h, evaporating the solvent and re-dissolving with DCM, washing twice with saturated NaHCO 3 , washing once with saturated brine, drying with anhydrous Na 2 SO 4 , and rotary drying. Without further column chromatography purification, it can be directly used for nucleic acid synthesis after dilution with anhydrous acetonitrile at a concentration of 0.05 g/mL. After solid phase synthesis, the corresponding Gal(N/O), Glu(N/O), and Man(N/O) conjugates can be obtained through further separation and purification.
  • FIG. 5 The flow chart of the liquid-solution convergence method for synthesizing G3-ON is shown in Figure 5.
  • a linker 1 with an amino terminus is synthesized using a solid phase synthesizer to obtain intermediate 1a for subsequent connection.
  • This intermediate is then cut with concentrated ammonia water at 60°C for 12 hours.
  • the supernatant is taken, the CPG powder is washed with pure water, the liquid is concentrated to remove ammonia, and then separated by Gilson preparative HPLC.
  • the flow chart of the solution convergence method for synthesizing the Cy5.5-G3-ON fluorescent marker is shown in FIG6 .
  • This method replaces the structure of amino linker 2 and uses 3′NH CPG.
  • the linker is programmed to be connected to the end of the nucleic acid chain, in the case of DMT-OFF, the intermediate 2a exposes the amino terminus.
  • the CPG powder is directly reacted with 5 times the amount of G3Ac-NHS active ester in a 1M TEAB solution environment for 5 hours to obtain intermediate 2b.
  • the same amino cutting and liquid phase separation methods as mentioned above are used to obtain intermediate 2c with exposed 3′NH (yield 25%).
  • intermediate 2c is reacted with 5 times the amount of Cy 5.5 active ester in a borate buffer system at pH 8 for 4 hours to obtain the final product.
  • the coupling product is further subjected to liquid phase purification (gradient elution method: ACN/0.05M TEAB; 0min-10% ACN, 30min-80% ACN; 4mL/min) to obtain the target pure Cy5.5-G3-ON fluorescent marker (total yield 20%), which is then lyophilized for later use.
  • the sequence of the antisense nucleic acid for CT102 MOE5 is: Tes m Ces m Ces Tes m Ces m C S G S G S A S G S m C S m C S A S G S A S m CesTesTes m CesAe;
  • m C represents 5-methylcytosine modification (5mC); s represents phosphorothioate modification (PS); e represents The structures are shown below:
  • the antisense nucleic acid sugar conjugate is selected from the following compounds:
  • the original sequence CT102 has relatively excellent resistance to enzymatic hydrolysis and serum stability due to its full thiochemical modification, and it has not been completely degraded in serum for 8 days (Figure 7).
  • the stability of the modified product based on this is more prominent, and the nuclease resistance is further enhanced. There is little degradation at all time points in the experiment.
  • the modified product already has strong serum stability, and the conjugate based on this has no obvious gain in serum stability.
  • a special phenomenon was found in this experiment that G3-CT102 MOE5 was significantly degraded, and a clear band appeared below the original chain.
  • CT102 MOE5 By adding CT102 MOE5 for comparison, it was found that this band was CT102 MOE5 , suggesting that G3-CT102 MOE5 will gradually fall off the G3 conjugated structure in serum, exposing CT102 MOE5 to continue to play an active role. This phenomenon was not found in the other conjugates, which may be related to the size of the conjugated group. The larger structure group is easily recognized and degraded by the enzyme.
  • GenOpti solution to a sterile enzyme-free EP tube, then add 10 ⁇ L of 200 ⁇ M ASO, 1.2 ⁇ L of 50 mM DNCA solution and 1.2 ⁇ L of 50 mM CLD solution in sequence, and then add 0.6 ⁇ L of 2 mM DSPE-PEG solution, make up the remaining GenOpti solution to 200 ⁇ L, and ultrasonicate at 50°C for 20 minutes before use.
  • HepG2/Huh7 cells were plated into 12-well plates at 100,000 cells/well and 50,000 cells/well, respectively, and cultured at 37°C. After 24 hours of culture, transfection was performed, wherein the nucleic acid concentration was 100 nM and the volume of the solution in each well was 100 ⁇ L. After 4 hours of administration, the supernatant was removed, the cells to be tested were washed once with PBS, 200 ⁇ L of 0.25% trypsin was added to each well, and after digestion for 2 minutes, 600 ⁇ L of 10% DMEM medium was added to each well, the cells were digested and transferred to a centrifuge tube, and the supernatant was removed by centrifugation at 1000 rpm for 3 minutes.
  • both GalNAc conjugates and Glu conjugates can increase the cell uptake of drugs to a certain extent, which may be due to the presence of glycosyl receptors on the cell surface, which leads to more mediated endocytosis of drugs.
  • the improvement in uptake rate was more obvious in HepG2 cells.
  • Example 8 Surface morphology, particle size and zeta potential of antisense nucleic acid preparations
  • the Malvern Zetasizer Nano-ZS laser scattering particle size analyzer was used for potential particle size determination, and the data was analyzed and processed using ELS-8000 software. At the same time, 20 ⁇ L of the above preparation was taken and the sample was processed using the negative staining method.
  • the sample was added dropwise to the PARA membrane and covered with a layer of copper mesh transparent film. After 1-2 minutes, the film was removed and the edge liquid was removed with absorbent paper. Subsequently, the copper mesh was stained with 1% uranyl acetate for 1 minute, the copper mesh was washed twice with PBS, and examined using a JEM-1400Plus transmission electron microscope (JEOL, Japan).
  • the particles of the other preparation groups were spherical with smooth surfaces.
  • the surface of the particles in the G3-CT102 MOE5 preparation group was rough, and it was speculated that part of the G3 conjugated structure was exposed on the surface of the liposome.
  • IVIS SPECTRUM small animal in vivo imager
  • RA , RB and RE represent the absorbance of the experimental group, the group without transfection reagent and the blank control group, respectively.
  • Target gene silencing activity HepG2 and Huh7 cells were plated into 12-well plates at 100,000 cells/well and 50,000 cells/well, respectively, and cultured at 37°C for 24 hours before transfection.
  • RNA reverse transcription and detection The total RNA was added in an amount of 500 ng, and after adding 5 ⁇ L of enzyme-free water, it was placed in a PCR instrument at 70°C for 10 min; each component was configured according to the instructions of the kit. The PCR execution program was 42°C for 15 min, 95°C for 5 min; and it was stored at 4°C. Subsequently, 10 ⁇ L of the above cDNA was diluted 5 times with 40 ⁇ L of enzyme-free water, and real-time quantitative PCR (40 cycles) was performed according to the standard procedure.
  • the upstream and downstream primers of IGF1R were (5′-3′): ATC GTT CAT CCA AGG CTG TTAC, AGC AAT GAG ACC TGT GTG CCTG.
  • the upstream and downstream primers of the internal reference ( ⁇ -actin) were (5′-3′): CCA ACC GCG AGA AGA TGA, CCA GAG GCG TAC AGG GAT AG.
  • Gal-CT102 MOE5 The long-chain conjugated structure (Gal (N/O), Glu (N/O, Man (N/O)) is slightly less active than the short-chain conjugated structure (Gal, Glu, Man). Based on the above results, Gal-CT102 MOE5 , Glu-CT102 MOE5 , Man-CT102 MOE5 , and G3-CT102 MOE5 were determined as the optimal structures in vitro, and subsequent in vivo efficacy experiments at the animal level were carried out.
  • the saline group (Blank) and sorafenib were set as positive control groups, and DNCA/CLD/DSPE-PEG2000 (Mix-20/20/0.4/1) encapsulated CT102, its 2′-MOE modification group, and CT102 MOE5 four different glycosyl conjugate groups.
  • each group of mice was intraperitoneally injected with the substrate luciferin every 7 days for in vivo imaging detection, and the tumor progression of each group of mice was observed and compared. The mice were killed after 28 days, and the livers were taken for photographing and weighing (depending on the situation, it was decided whether the tumor mass was separated from the liver).
  • liver sections were made.
  • the intracellular IGF-IR protein was immunofluorescently stained and read to observe the inhibition of target expression.
  • Liver paraffin sections were made and the liver tissue was pathologically analyzed.
  • peripheral blood was taken from the mice, and plasma was separated for various blood biochemical tests that can characterize liver and kidney function.
  • the drug safety was analyzed in combination with the weight of the mice during the entire experimental period (Table 2).
  • sequences with anti-liver cancer activity reported in the literature, such as PHN01, PHN02, and PHN03 sequences (from the literature [Prog Biochem Biophys, 2002, 29 (2): 247-251.]), like CT102, PHN01, PHN02 and PHN03 sequences all target the first segment of the protein coding region (Coding Sequence, CDS) in the IGF1R mRNA sequence.
  • CDS Coding Sequence
  • Example 13 Differences between the novel antisense nucleic acid sequences and CT102 in HCC cell proliferation inhibition, target gene silencing and cell apoptosis
  • Each new antisense nucleic acid sequence (shown in Table 5) was ordered from Bio-Technology (Shanghai) Co., Ltd.
  • HepG2 cells were cultured in 12-well plates at a density of 1 ⁇ 10 5 cells/well for 18 hours, and then exposed to the above antisense nucleic acid preparations. After incubation for 24 hours, the medium was discarded, and then the cells were trypsinized according to the manufacturer's protocol, collected and stained with Annexin V-FITC/PI cell apoptosis detection kit, and the proportion of apoptotic cells was analyzed by flow cytometry.
  • Example 14 Antisense nucleic acid novel sequences PHN02 and PHN07 in A549, MCF-7, B-CPAP cell proliferation inhibition and target gene silencing activity
  • CCK-8 assay A549, MCF-7, and B-CPAP cells were plated into 96-well plates at 3,000 cells/well, 3,000 cells/well, and 6,000 cells/well, respectively, and cultured at 37°C for 24 hours before transfection using the same method as in Example 10;
  • Target gene silencing activity A549, MCF-7, and B-CPAP cells were plated into 12-well plates at 50,000 cells/well, 50,000 cells/well, and 60,000 cells/well, respectively, and cultured at 37° C. for 24 h before transfection using the same method as in Example 6.
  • Example 15 Differences between 2′-O-MOE modified PHN02 and CT102 and PHN02 in terms of cell proliferation inhibition and target gene silencing
  • CT102 and PHN02 were further modified to obtain sequences CT102 MOE5 and PHN02 MOE5 with 5 2′-O-MOE modifications at both ends and all cytosines being 5 m C.
  • the modified antisense nucleic acids were synthesized in the laboratory as shown in Table 6.
  • the cell proliferation inhibitory activity and target gene silencing activity were performed in the same manner as in Example 10.
  • Results The results showed ( Figure 17) that the anti-liver cancer effects of PHN02 MOE5 and CT102 MOE5 were basically the same, and the anti-liver cancer effects of PHN02 MOE5 and CT102 MOE were further improved than those of PHN02 and CT102.
  • the drug efficacy on the proliferation inhibition of HepG2 and Huh-7 cells and the IGF1R mRNA silencing effect were significantly improved compared with CT102.

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Abstract

L'invention concerne un conjugué glycosyle d'acide nucléique antisens, qui est formé par conjugaison covalente d'une molécule de sucre à l'extrémité 5' d'un acide nucléique antisens au moyen d'un bras de liaison. La pharmacodynamique in vitro montre que l'activité de prolifération cellulaire anti-tumorale et l'activité de silençage du gène cible de divers conjugués sont comparables à celles des chaînes d'acides nucléiques antisens non conjuguées. Après conjugaison, le taux d'absorption de médicament est amélioré, ce qui démontre une activité inhibitrice du cancer du foie in vivo plus élevée. Glu-CT102MOE5 présente l'effet le plus significatif. Les activités in vitro et in vivo des acides nucléiques antisens PHN02 et PHN07 sont importantes à celles de CT102. Après une autre modification chimique, la capacité anti-tumorale de PHN02MOE5 est comparable à celle de CT102MOE5. Le conjugué glycosyle d'acide nucléique antisens peut être utilisé pour le traitement du cancer du foie.
PCT/CN2023/121152 2022-09-29 2023-09-25 Conjugué glycosyle d'acide nucléique antisens, son procédé de préparation et son utilisation dans le traitement du cancer du foie WO2024067500A1 (fr)

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