An oligonucleotide drug preparation for treating liver cancer and a preparation method and application thereof

Abstract

The application discloses an oligonucleotide drug preparation applied to liver cancer treatment and a preparation method and application thereof. The drug preparation is composed of an oligonucleotide drug, a carrier and a solvent, wherein the oligonucleotide drug is PHN02 ME , the oligonucleotide drug PHN02 ME adopts 5 cap modifications at two ends on the basis of a full thio sequence, and the carrier is composed of nucleotide phospholipid TPS or CPS, cationic lipid CLDA and auxiliary lipid DSPE-PEG. The application obtains a new liver cancer resistant drug preparation with great clinical application potential through preparation proportion adjustment and preparation solvent optimization. After low-dose and low-frequency intravenous administration of the preparation to mice, the growth of liver cancer in the mice is significantly inhibited, and the preparation shows ideal sustained release / long-acting anticancer effect, and the drug efficacy is better than that of a CT102 / DNCA / CLD / PEG preparation. The application lays a foundation for the wide clinical application of the liver cancer resistant antisense nucleic acid drug, and also has wide application prospects in the field of gene therapy.

Description

Technical Field

[0001] This invention relates to an oligonucleotide drug formulation for the treatment of liver cancer, its preparation method, and its application. This invention belongs to the field of biomedical technology. Background Technology

[0002] Primary liver cancer is currently the fourth most common malignant tumor and the second leading cause of cancer death in my country, seriously threatening the lives and health of the Chinese people. Primary liver cancer mainly includes three different pathological types: hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), and combined hepatocellular-cholangiocarcinoma (cHCC-CCA). These three types differ significantly in their pathogenesis, biological behavior, pathological histology, treatment methods, and prognosis. HCC accounts for 75%–85% of cases, while ICC accounts for 10%–15%.

[0003] Because HCC has an insidious onset, less than 30% of HCC patients are suitable for radical treatment at the time of initial diagnosis. Systemic antitumor therapy plays an important role in the treatment of intermediate and advanced liver cancer, controlling disease progression and prolonging patient survival.

[0004] Nucleic acid drugs are currently a research hotspot, used for regulating physiological processes at the gene level and treating diseases. Delivery systems are crucial for nucleic acid drugs to achieve stable targeted efficacy and have become the biggest obstacle affecting their drug development. LNP encapsulation or GalNAc (N-acetylgalactosamine) conjugation are commonly used delivery methods in nucleic acid, especially siRNA, drug development. Both are based on liver-specific biological mechanisms for targeted delivery, and these methods are patented in Europe and the United States.

[0005] In the development of drugs for liver cancer (HCC), antibody drugs primarily target PD-1 / PD-L11 and CTLA-42, while chemical drugs target VEGFR3 and BRAF4 proteins. Due to the high rate of drug resistance in HCC, the above therapies suffer from low specificity and short half-lives, leading to unsatisfactory prognoses. Antisense nucleic acids or siRNAs have therapeutic effects on HCC. TKM-080301, an anti-liver cancer siRNA drug targeting Polo-like kinase 1 (PLK1) developed by Arbutus Biopharma, has completed clinical trials. The starting dose was 0.3 mg / kg, and the recommended dose was 0.6 mg / kg. The median overall survival was 7.5 months (compared to 6.5 months for sorafenib), and no clinically significant higher activity was observed. The small activating RNA (saRNA) drug MTL-CEBPA upregulates transcription factor C / EBP-α (CCAAT / enhancer-binding protein α). In a rat model of cirrhosis with multifocal liver tumors, three intravenous injections (20 nM) reduced tumor burden by 80%. Clinical trials showed good safety and potential synergistic effects with tyrosine kinase inhibitors. Sodium phosphothiodeoxy oligonucleotide CT102 targets the mRNA of human insulin-like growth factor receptor 1 (IGF1R) and can induce tumor cell apoptosis or differentiation for the treatment of HCC. Phase II clinical trials have begun in China. Although the clinical dosage (~5 MPk) is moderate, significant toxic side effects have still occurred. Summary of the Invention

[0006] The purpose of this invention is to provide an oligonucleotide drug formulation for the treatment of liver cancer, its preparation method, and its application.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention discloses an oligonucleotide drug formulation for the treatment of liver cancer, wherein the drug formulation comprises an oligonucleotide drug, a carrier, and a solvent, wherein the oligonucleotide drug is PHNO2. ME The sequence is 5'-GAGAAACAGGAGCCCCCACA-3', and the oligonucleotide drug PHN02 is described. ME Based on the fully thiolated sequence, five caps were applied to each end of the sequence. The selected cap modification structure was 2′-O-MOE, as shown below:

[0009]

[0010] The carrier is composed of nucleoside phospholipids TPS or CPS, cationic lipid CLDA, and auxiliary lipid DSPE-PEG. The structural formulas of TPS, CPS, CLDA, and DSPE-PEG are shown below:

[0011]

[0012] Where B is cytosine or thymine, and R1 is C 18 H 35 R2 is C 16 H 33 .

[0013] Preferably, the molar ratio of the nucleoside phospholipid TPS or CPS, the cationic lipid CLDA, and the oligonucleotide drug is TPS or CPS:CLDA:oligonucleotide drug = 20:20:1, 20:40:1, 25:35:1, 30:30:1, 35:35:1, or 40:40:1, and the amount of auxiliary lipid DSPE-PEG used is 1% of the total weight of TPS or CPS and cationic lipid CLDA.

[0014] Preferably, the solvent is GenOpti or contains Ca. 2+ PBS.

[0015] Preferably, Ca is added. 2+ The phosphate concentration in the PBS was 5.95 mM, and the Ca2+ concentration was 100 mg / L. 2+ A concentration of 0.2 mM or a phosphate concentration of 11.9 mM, Ca 2+ The concentration is 1 mM.

[0016] Furthermore, the present invention also provides a method for preparing the aforementioned oligonucleotide drug formulation, comprising the following steps:

[0017] (1) The oligonucleotide drug PHN02 ME Prepare stock solutions with concentrations of 0.05 mM to 10 mM using enzyme-free water; prepare stock solutions with concentrations of 1 mM to 1 M using enzyme-free water for CaCl2; prepare stock solutions with concentrations of 1 mM to 100 mM using anhydrous ethanol for TPS and CLDA; and prepare stock solutions with concentrations of 0.1 mM to 50 mM using anhydrous ethanol for DSPE-PEG.

[0018] (2) Add oligonucleotide drug PHN02 to the centrifuge tube. ME The mother liquor, then add half a volume of GenOpti solution or add Ca 2+ Mix with PBS;

[0019] (3) Add TPS, CLDA, and DSPE-PEG anhydrous ethanol mother liquor in sequence close to the liquid surface;

[0020] (4) Replenish the remaining half volume of GenOpti solution or add Ca. 2+ PBS;

[0021] (5) 50℃, 4KHz ultrasound for 20min.

[0022] Furthermore, the present invention also proposes the application of the aforementioned oligonucleotide pharmaceutical formulation in the preparation of drugs for treating primary liver cancer.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] This invention utilizes nucleoside phospholipids combined with cationic lipid materials, along with auxiliary lipid materials. Through formulation ratio screening and solvent optimization, formulation conditions suitable for anti-hepatocellular carcinoma antisense nucleic acid encapsulation were obtained, encapsulating PHNO2. ME The resulting formulation has simple and controllable components, and exhibits highly effective and long-lasting anti-hepatocellular carcinoma effects in an orthotopic hepatocellular carcinoma mouse model. It can restore liver damage caused by hepatocellular carcinoma and has a high safety profile. Attached Figure Description

[0025] Figure 1 PHN02 ME Flowchart of the preparation method for / TPS(or CPS) / CLDA / PEG formulation;

[0026] Figure 2 For different ratios of TPS / CLDA / PHN02 ME The inhibitory effect of the formulation on the proliferation of three types of liver cancer cells;

[0027] Blank: blank solvent control; D:C represents DNCA:CLD-encapsulated formulation; the remaining groups are TPS:CLDA-encapsulated formulations; PHN02 ME Dosage concentration: 50 nM;

[0028] Figure 3 For different ratios of CPS / CLDA / PHN02 ME The inhibitory effect of the formulation on the proliferation of three types of liver cancer cells;

[0029] Blank: blank solvent control; D: C represents DNCA: CLD-encapsulated formulation; the remaining groups are CPS: CLD-encapsulated formulations; PHN02 ME Dosage concentration: 50 nM;

[0030] Figure 4 and Figure 5 PHN02 ME The inhibitory effect of nucleoside phospholipid preparations on the growth of in situ hepatocellular carcinoma in BALB / c nude mice (n=6);

[0031] Figure 4In the diagram, A. Experimental flowchart; B. Relative tumor growth fold of each group of mice (vs. fluorescence intensity of the D0 tumor site); C. Tumor fluorescence value of each group of mice; D. Blank group, PHN02 ME TPS:CLDA (1:20:20) group mice tumor D21 imaging image and D28 liver photograph;

[0032] Figure 5 In the table, A. Photographs of mouse tumors in each group; B. Tumor weight in each group of mice; C. IGF1R mRNA expression level in mouse tumors in each group of mice; D. IGF1R protein staining (brown) results of tumor sections in each group of mice.

[0033] Figure 6 and Figure 7 PHN02 ME The safety profile of nucleoside phospholipid preparations in a BALB / c nude mouse orthotopic liver cancer model.

[0034] Figure 6 In the following data: A. Body weight of mice in each group; B. Plasma alanine aminotransferase (ALT) level of mice in each group; C. Plasma aspartate aminotransferase (AST) level of mice in each group; D. Plasma total bilirubin level of mice in each group; E. Plasma urea level of mice in each group; F. Plasma creatinine level of mice in each group.

[0035] Figure 7 In the study, the following parameters were considered: A. Plasma IL-6 levels in each group of mice; B. Plasma IFN-γ levels in each group of mice; C. Plasma IL-1β levels in each group of mice; D. Plasma TNF-α levels in each group of mice; E. The levels of the formulations in group 3 (PHN02ME / TPS / CLDA, 1 / 20 / 20) and group 7 (PHN02...). ME HE staining results of mouse liver sections (TPS / CLDA, 1 / 30 / 30, PBS / Ca-1); F. Group 3 formulation (PHN02) ME / TPS / CLDA, 1 / 20 / 20) and Group 7 formulation (PHN02) ME HE staining results of mouse kidney sections (TPS / CLDA, 1 / 30 / 30, PBS / Ca-1);

[0036] Figure 8 and Figure 9 PHN02 ME The inhibitory effect of nucleoside phospholipid preparations (in different solvents) on the growth of orthotopic liver cancer in NPG mice (n=5-7);

[0037] Figure 8 In the diagram, A. Experimental flowchart; B. Relative tumor growth fold of each group of mice (vs. autologous D0 tumor fluorescence intensity); C. Tumor fluorescence value of each group of mice; D. Blank group, PHN02 METumor D26 imaging images and liver photographs of mice in the / TPS / CLDA(1 / 20 / 20) group;

[0038] Figure 9 In the table, A. Photographs of mouse tumors in each group; B. Tumor weight in each group of mice; C. IGF1R mRNA expression level in mouse tumors in each group of mice; D. IGF1R protein staining (brown) results of tumor sections in each group of mice.

[0039] Figure 10 and Figure 11 PHN02 ME The safety profile of nucleoside phospholipid preparations (in different solvents) in an NPG mouse orthotopic liver cancer model.

[0040] Figure 10 In the following data: A. Body weight of mice in each group; B. Plasma alanine aminotransferase (ALT) level of mice in each group; C. Plasma aspartate aminotransferase (AST) level of mice in each group; D. Plasma total bilirubin level of mice in each group; E. Plasma urea level of mice in each group; F. Plasma creatinine level of mice in each group.

[0041] Figure 11 In the study, A. Plasma IL-6 levels in each group of mice; B. Plasma IFN-γ levels in each group of mice; C. Plasma IL-1β levels in each group of mice; D. Plasma TNF-α levels in each group of mice; E. The level of the third formulation (PHN02) ME / TPS / CLDA, 1 / 20 / 20) and Group 5 formulation (PHN02) ME HE staining results of mouse liver sections (TPS / CLDA, 1 / 30 / 30, PBS / Ca-1); F. Group 3 formulation (PHN02) ME / TPS / CLDA, 1 / 20 / 20) and Group 5 formulation (PHN02) ME HE staining results of mouse kidney sections (TPS / CLDA, 1 / 30 / 30, PBS / Ca-1). Detailed Implementation

[0042] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description of the specific embodiments. However, the embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0043] Example 1

[0044] This example mainly illustrates the relationship between TPS, CLDA, DSPE-PEG, and the oligonucleotide drug PHN02. METhe process for preparing a drug formulation by mixing is shown in the flowchart below. Figure 1 As shown.

[0045] Materials and Methods:

[0046] (1) The PHN02 sequence (5'-GAGAAACAGGAGCCCCCACA-3') is from the literature [Prog BiochemBiophys,2002,29(2):247-251]. Based on the fully thiolated sequence, it is modified by adding 5 caps at each end. The selected structure for the cap modification is 2′-O-MOE, and its structure is shown below:

[0047]

[0048] (2) Based on the needs of different types of experiments, the oligonucleotide drug PHN02 ME Prepare stock solutions with concentrations of 0.05 mM to 10 mM using enzyme-free water; prepare stock solutions with concentrations of 1 mM to 1 M using enzyme-free water for CaCl2; prepare stock solutions with concentrations of 1 mM to 100 mM using anhydrous ethanol for TPS and CLDA; and prepare stock solutions with concentrations of 0.1 mM to 50 mM using anhydrous ethanol for DSPE-PEG.

[0049] The structural formulas of TPS, CPS, CLDA, and DSPE-PEG are shown below:

[0050]

[0051] Where B is cytosine or thymine, and R1 is C 18 H 35 R2 is C 16 H 33 .

[0052] (3) Add oligonucleotide drug PHN02 to the centrifuge tube. ME The mother liquor, then add half a volume of GenOpti solution or add Ca 2+ Mix with PBS; use GenOpti solution or add Ca 2+ The amount of PBS used can be determined according to the actual concentration of the drug solution to be prepared;

[0053] (4) Add TPS, CLDA, and DSPE-PEG anhydrous ethanol mother liquor in sequence close to the liquid surface;

[0054] (5) Replenish the remaining half volume of GenOpti solution or add Ca. 2+ PBS;

[0055] (6) 50℃, 4KHz ultrasound for 20min.

[0056] Add Ca 2+ The phosphate concentration in the PBS was 5.95 mM, and the Ca2+ concentration was 100 mg / L. 2+ A concentration of 0.2 mM or a phosphate concentration of 11.9 mM, Ca 2+ The concentration is 1 mM.

[0057] Example 2

[0058] This embodiment mainly illustrates the oligonucleotide drug (ASO) PHN02 ME The formulation inhibits the proliferation of liver cancer cells after administration.

[0059] Materials and Methods: Ten formulation groups were set up as shown in Table 1. The optimal formulation ratio of TPS or CPS combined with cationic liposome CLDA-encapsulated oligonucleotide drugs was screened using the CCK8 assay. Each group was supplemented with DSPE-PEG2000 at 1% of the weight of the liposomes (TPS or CPS+CLDA). HepG2 and HepG2-Luc cells were seeded at 10,000 cells per well, and Huh-7 cells at 6,000 cells per well in 96-well plates. After 24 hours, samples were prepared according to the formulation method in Example 1, with an oligonucleotide concentration of 100 nM and a solution volume of 200 μL, and then administered. After 48 hours, CCK-8 substrate was added to each well, and the cells were incubated at 37°C in the dark for 1 hour. The absorbance at 450 nm was measured using a Microplate Reader (Molecular Devices, California, USA), and cell viability was calculated using the following formula:

[0060] Cell Viability = (R A -R E ):(R B -R E )×100%

[0061] Note: R A R B R E The absorbance values ​​represent the experimental group, the group without transfection reagent, and the blank control group, respectively.

[0062] Table 1 shows the results of TPS or CPS combined with cationic lipid material CLDA-encapsulated PHNO2 in the CCK-8 experiment. ME formulation ratio

[0063] Serial Number TPS (or CPS): CLDA: ASO Serial Number TPS (or CPS): CLDA: ASO 1 20：20：1 6 30：30：1 2 20：40：1 7 35：25：1 3 25：25：1 8 35：35：1 4 25：35：1 9 40：20：1 5 30：20：1 10 40：40：1

[0064] Result: As Figure 2 and Figure 3As shown, all formulations exhibited significant inhibitory effects on HCC tumor proliferation. Among them, the efficacy was more pronounced when the molar ratio of TPS (or CPS):CLDA:ASO was 20:40:1, 25:35:1, 30:30:1, 35:35:1, and 40:40:1. Furthermore, when the molar ratio of TPS (or CPS):CLDA:ASO was 20:20:1, relatively good efficacy could be maintained even with a smaller amount of lipid material.

[0065] Example 3

[0066] This embodiment mainly explains PHN02 ME Efficacy and safety of / TPS (or CPS) / CLDA / PEG formulation in BALB / c nude mice for hepatocellular carcinoma.

[0067] Materials and Methods: BALB / c nude mice were selected as an animal model. A mutation in the recessive gene Foxn1 (forkheadbox N1) on chromosome 11 causes abnormal hair growth and development (absence of hair), accompanied by congenital thymic hypoplasia, T cell deficiency, normal B cells, and slightly elevated NK cells. While commonly used as recipients of tumor xenografts, their enhanced NK cell activity and normal humoral immunity prevent the transplantation of human hematopoietic stem cells. HepG2-luc cells stably transfected with the luciferase gene were cultured and then inoculated into the axillae of several BALB / c nude mice. When the subcutaneous xenograft reached approximately 10 mm in diameter, fluorescence intensity was confirmed by photographing. The xenograft was then removed, the tumor nodule was cut open, necrotic tissue in the center was removed, and small, rice-grain-sized tumor pieces (approximately 1 mm in diameter) were cut and sealed onto the liver of BALB / c nude mice using a special adhesive. After successful modeling, intraperitoneal injection of its substrate, luciferin D, resulted in luminescence within minutes to tens of minutes. The intensity of the luminescence was linearly correlated with the number of live cells, facilitating the observation of tumor progression in live mice. Specific experimental protocols are shown in Table 2. Figure 4 A.

[0068] Table 2 shows PHN02 ME Efficacy and safety study protocol of / TPS (or CPS) / CLDA / PEG formulation in BALB / c nude mice for hepatocellular carcinoma.

[0069]

[0070] Results: The second group of formulations (CT102:DNCA:CLD = 1:20:20), serving as a positive control, significantly inhibited the growth of liver cancer; the third group of formulations (PHN02)... ME The TPS:CLDA ratio of 1:20:20 showed the best anti-liver cancer effect, with no tumor growth rebound within 28 days, which was superior to the second group of preparations. Figure 4 B, C); Group 7 formulation (PHN02) ME The PBS / Ca-1 ratio (TPS:CLDA = 1:30:30, PBS / Ca-1) also showed significant anti-hepatocellular carcinoma activity, exhibiting tumor inhibition comparable to the third group of preparations on day 21. However, tumor growth rebounded afterward, suggesting that in this animal model, adjusting the dosing frequency may lead to even better efficacy. Figure 4 (B, C). In addition, all mice in the drug-treated groups survived to the experimental endpoint; two mice in the Blank group died on D26:D27 due to excessive tumor growth; all mice in the fourth group died on D3 / D4 after the first administration of the drug, and the cause is yet to be determined.

[0071] At the experimental endpoint, the mice were dissected, and a series of organ-level, tissue-level, and molecular-level analyses were performed. The results showed that the livers of mice in the antisense nucleic acid preparation group were smoother, with smaller tumors and no significant nodules; the Blank group had larger, more diffuse tumors with more nodules. Figure 4 D); Dissection to remove the tumor mass and photograph it ( Figure 5 A) Weigh ( Figure 5 B) The tumors in the antisense nucleic acid preparation group were all smaller than those in the Blank group, among which, the preparation in group 3 (PHN02) ME (TPS:CLDA, 1:20:20), Group 6 formulation (PHN02) ME CPS:CLDA, 1:25:35), Group 7 formulation (PHN02) ME The tumor weight (TPS:CLDA, 1:30:30, PBS / Ca-1) was significantly different from that of the Blank group (P value is shown in the figure); immunohistochemical experiments were used to detect the expression level of IGF1R protein in tumor sections. Figure 5 D), it was found that the IGF1R protein level was significantly lower in all formulation groups compared to the Blank group, with the third formulation (PHN02) showing the lowest level. ME The TPS:CLDA (1:20:20) group of mice had the lowest level of IGF1R protein in tumors; RT-qPCR results showed that all preparations could significantly silence IGF1R mRNA in tumor tissues (P value is shown in the figure), and the third group of preparations had the strongest silencing effect.

[0072] Throughout the animal experiment, the body weight of mice in each group remained relatively stable. In the later stages of the disease, the mice's condition deteriorated, and their body weight began to decrease. Overall, the mice in the formulation-treated group were in better condition than the Blank group, and their body weight loss was slower in the Blank group. Figure 6 A). The occurrence of liver cancer resulted in elevated ALT, AST, and TBIL levels in mice, indicating liver damage. The third formulation (PHN02) was used to treat this. ME (TPS:CLDA = 1:20:20) and Group 7 formulation (PHN02) MEAfter treatment with TPS:CLDA = 1:30:30 (PBS / Ca-1), ALT, AST, and TBIL levels significantly decreased, indicating an improvement in liver damage. Figure 6 (BD); The occurrence of liver cancer did not significantly affect the UREA and CREA values ​​in mice, and there were no significant differences in UREA and CREA values ​​among the different groups after administration of the formulation, indicating that the formulation had no significant nephrotoxicity. Figure 6 E, F). ELISA was used to detect inflammatory factors IL-6, IFN-γ, IL-1β, and TNF-α in mouse blood. The results showed that there were no significant changes in any of the inflammatory factors after administration of the formulations in each group, indicating that the formulations had low immunogenicity and no risk of inducing an immune storm. Figure 7 AD). HE staining results of liver and kidney sections indicated that the third formulation (PHN02)... ME (TPS:CLDA, 1:20:20) and Group 7 formulation (PHN02) ME (TPS:CLDA, 1:30:30, PBS:Ca-1) Mouse liver and kidney cells showed normal morphology and no significant pathological changes caused by the preparation. Figure 7 E, F).

[0073] Example 4

[0074] This embodiment mainly explains PHN02 ME Efficacy and safety of the / TPS(or CPS) / CLDA / PEG formulation for hepatocellular carcinoma in NPG mice.

[0075] Materials and Methods: To further observe the effects of rapid tumor growth to a large volume in PHN02 tumors... METhe anti-hepatocellular carcinoma effect of the / TPS (or CPS) / CLDA / PEG formulation was investigated by changing the animal model to NPG mice. This strain of mice is the most immunodeficient mouse model in the world to date. Its Prkdc (protein kinase DNA-activated catalytic) gene mutation results in the absence of both T and B cells, manifesting as severe combined immune deficiency (SCID) of cellular and humoral immunity. Furthermore, since the γ chain of the IL2 receptor protein is a key component of the high-affinity receptors for the cytokines IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, which have important immune functions, and is essential for the receptor to transmit these cytokine signals, gene knockout severely reduces the body's immune function, especially the activity of NK cells is almost lost. After expanding the culture of HepG2-luc cells stably transfected with the luciferase gene, they were inoculated into the axillae of several BALB / c nude mice. When the subcutaneous xenografts reached approximately 10 mm in diameter, the fluorescence intensity was confirmed by photographing. The xenografts were then removed, the tumor nodules were cut open, necrotic tissue in the center was removed, and small, rice-grain-sized tumor fragments (approximately 1 mm in diameter) were cut and sealed onto the livers of NPG mice using a special adhesive. After successful modeling, intraperitoneal injection of the substrate luciferin D-luciferin resulted in luminescence within minutes to tens of minutes. The intensity of the luminescence was linearly correlated with the number of live cells, facilitating the observation of tumor progression in live mice. The specific experimental protocol is shown in Table 3.

[0076] Table 3 shows PHN02 ME Efficacy and safety study of / TPS (or CPS) / CLDA / PEG formulations in NPG mice for hepatocellular carcinoma.

[0077]

[0078] Results: The second group of formulations (CT102:DNCA:CLD = 1:20:20), serving as a positive control, significantly inhibited the growth of liver cancer; the third group of formulations (PHN02)... ME (TPS:CLDA = 1:20:20) and Group 5 formulation (PHN02) ME The TPS:CLDA ratio of 1:30:30 (PBS / Ca-1) showed the best anti-hepatocellular carcinoma effect, superior to the second group of formulations; the third and fifth group formulations exhibited relatively ideal sustained-release and long-acting anti-cancer effects. Figure 8 B, C), further inhibited tumor growth after D21 (specifically, the tumor growth fold D26: D21 < 1); analysis of D26 data ( Figure 8A) The tumor growth rate of mice in the 3rd, 4th and 5th formulation groups was significantly different from that in the Blank group (P≤0.05), and the total flux of mice in the 5th formulation group was significantly different from that in the Blank group (P≤0.05).

[0079] At the experimental endpoint, mice were dissected, and their livers (including tumors) were removed and photographed. The liver tumors in the antisense nucleic acid preparation group were smaller; the tumors in the Blank group were larger. Figure 8 D); The tumor mass was removed and photographed. Figure 9 A) Weigh ( Figure 9 B), the tumors in the antisense nucleic acid preparation group were all smaller than those in the Blank group, among which, the preparation in group 3 (PHN02) ME TPS:CLDA = 1:20:20), Group 5 formulation (PHN02) ME TPS:CLDA = 1:30:30, PBS / Ca-1), Group 6 formulation (PHN02) ME The tumor weight of the TPS:CLDA group (TPS:CLDA = 1:30:30, PBS / Ca-2) was significantly different from that of the Blank group (P≤0.001, P≤0.001, P≤0.01); RT-qPCR results showed that all preparations could significantly silence IGF1R mRNA in tumor tissue (P values ​​are shown in the table). Figure 9 C) The silencing effects of the formulations in each group were comparable; immunohistochemical experiments detected the expression level of IGF1R protein (brown) in tumor sections, demonstrating that the amount of IGF1R protein in tumor tissue was significantly decreased in each formulation group compared with the Blank group. Figure 9 D).

[0080] Throughout the entire animal experiment period, mice in the Blank group were in poorer condition and experienced weight loss in the later stages of disease development, while mice in the formulation administration group were in better condition and did not show significant weight loss. Figure 10 A). The occurrence of liver cancer resulted in elevated ALT, AST, and TBIL levels in mice, indicating liver damage. The third formulation (PHN02) was used to treat this. ME (TPS:CLDA = 1:20:20) and Group 5 formulation (PHN02) ME After treatment with TPS:CLDA = 1:30:30 (PBS / Ca-1), ALT, AST, and TBIL levels significantly decreased, indicating an improvement in liver damage. Figure 10 (BD); The occurrence of liver cancer did not significantly affect the UREA and CREA values ​​in mice, and there were no significant differences in UREA and CREA values ​​among the different groups after administration of the formulation, indicating that the formulation had no significant nephrotoxicity. Figure 10E, F). ELISA was used to detect inflammatory factors IL-6, IFN-γ, IL-1β, and TNF-α in mouse blood. The results showed that after administration of the formulations in each group, there were no significant changes in any of the inflammatory factors, indicating that the formulations had low immunogenicity and no risk of inducing an immune storm. Figure 11 AD). HE staining results of liver and kidney sections indicated that the third formulation (PHN02)... ME (TPS:CLDA = 1:20:20) and Group 5 formulation (PHN02) ME (TPS:CLDA = 1:30:30, PBS / Ca-1) Mouse liver and kidney cells showed normal morphology and no significant pathological changes caused by the preparation. Figure 11 E, F).

[0081] The information shown and described in detail herein is sufficient to achieve the above-described objectives of the invention. Therefore, the preferred embodiments of the invention represent the subject matter of the invention, which is broadly covered by the invention. The scope of the invention fully encompasses other embodiments that will be obvious to those skilled in the art, and therefore, the scope of the invention is not limited by anything other than the appended claims, wherein, unless expressly stated otherwise, the singular form of an element does not mean "one and only," but rather "one or more." All structural, compositional, and functional equivalents of the foregoing preferred embodiments and additional embodiments known to those skilled in the art are therefore incorporated herein by reference and are intended to be covered by the claims of the invention.

[0082] Furthermore, no specific device or method is required to express each problem solved by this invention, as they are all included within the scope of the claims. Additionally, regardless of whether all parts, components, or method steps disclosed in this invention are expressly described in the claims, they are not made public. However, it will be apparent to those skilled in the art that various changes and modifications can be made in form, reagents, and synthetic details without departing from the spirit and scope of the invention as set forth in the appended claims.

Claims

1. An oligonucleotide drug formulation for the treatment of liver cancer, characterized in that, The pharmaceutical formulation comprises an oligonucleotide drug, a carrier, and a solvent, wherein the oligonucleotide drug is PHNO2. ME The sequence is 5'-GAGAAACAGGAGCCCCCACA-3', and the oligonucleotide drug PHN02 is described. ME Based on the fully thiolated sequence, five caps were applied to each end of the sequence. The selected cap modification structure was 2ʹ- O -MOE, its structure is as follows: ； The carrier is composed of nucleoside phospholipid TPS, cationic lipid CLDA, and auxiliary lipid DSPE-PEG, and the structural formulas of TPS, CLDA, and DSPE-PEG are shown below: ； Where B is thymine and R1 is C 18 H 35 R2 is C 16 H 33 ; The molar ratio of the nucleoside phospholipid TPS, the cationic lipid CLDA, and the oligonucleotide drug is TPS:CLDA:oligonucleotide drug = 20:20:1, and the amount of the auxiliary lipid DSPE-PEG used is 1% of the total weight of TPS and the cationic lipid CLDA. The solvent mentioned above is one with added Ca. 2+ PBS; with added Ca 2+ The phosphate concentration in the PBS was 5.95 mM, and the Ca2+ concentration was 100 mg / L. 2+ The concentration is 0.2 mM.

2. A method for preparing the oligonucleotide pharmaceutical formulation of claim 1, characterized in that, Includes the following steps: (1) The oligonucleotide drug PHN02 ME Prepare a stock solution with a concentration of 0.05 mM to 10 mM using enzyme-free water; prepare a stock solution with a concentration of 1 mM to 1 M using enzyme-free water for CaCl2; prepare a stock solution with a concentration of 1 mM to 100 mM using anhydrous ethanol for TPS and CLDA; and prepare a stock solution with a concentration of 0.1 mM to 50 mM using anhydrous ethanol for DSPE-PEG. (2) Add the oligonucleotide drug PHN02 to the centrifuge tube. ME The mother liquor, then add half the volume of added Ca. 2+ Mix with PBS; (3) Add TPS, CLDA, and DSPE-PEG anhydrous ethanol mother liquor in sequence close to the liquid surface; (4) Add Ca to complete the other half of the volume 2+ PBS; (5) 50℃, 4KHz ultrasound for 20min.

3. The use of the oligonucleotide pharmaceutical formulation according to claim 1 in the preparation of a drug for treating primary liver cancer.

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