Thiothioate oligonucleotide injection and preparation method thereof

Abstract

The present disclosure provides a thiolated oligonucleotide injection and a method for preparing the same. This preparation comprises an aqueous solution of a thiolated antisense oligonucleotide targeting the IGF1R gene. The method for preparing this preparation comprises preparing an aqueous solution comprising a thiolated antisense oligonucleotide targeting the IGF1R gene and optionally adding an injectable pharmaceutically acceptable carrier. The present disclosure also provides a lyophilized preparation comprising an aqueous solution of a thiolated antisense oligonucleotide targeting the IGF1R gene, and a method for preparing the lyophilized preparation. Further, the present disclosure also provides the use of the above-mentioned injection preparation and lyophilized preparation in the treatment of cancers associated with the IGF1R gene. By utilizing the method disclosed in the present disclosure, the stability of the oligonucleotide is increased, and the expression of the targeted gene can be effectively inhibited, thereby inhibiting the growth of the tumor.

Description

Technical Field

[0001] The present disclosure relates to the field of nucleic acid pharmaceutical preparations, and in particular to an injection containing thiolated oligonucleotides and a preparation method thereof. Background Art

[0002] Oligonucleotide is a general term for a class of short-chain nucleotides with less than 30 bases (including deoxyribonucleotides or ribonucleotides). Antisense oligonucleotides are used to inhibit RNA fragments and prevent them from being translated into proteins. They play a certain positive role in the treatment of diseases such as cancer.

[0003] Antisense oligodeoxynucleotides (ASOs) are one of the methods for targeted regulation of the IGF1R signaling pathway. They can form double strands by base pairing with specific sequences of target mRNA, thereby activating RNase H to degrade mRNA or prevent RNA from translating into proteins, thereby inhibiting the expression of the target gene. So far, two antisense nucleic acid drugs have been approved for marketing internationally (Vitravene TM and ).

[0004] Since antisense oligonucleotides have poor stability, they cannot play a good therapeutic role. Therefore, there is an urgent need in the art for stable antisense oligonucleotide preparations to increase their therapeutic effects. Summary of the invention

[0005] In view of the above problems, the present disclosure provides a lyophilized oligonucleotide preparation. This antisense oligonucleotide preparation can not only improve the serum stability of the antisense oligonucleotide, but also be effectively delivered to the liver, thereby efficiently inhibiting the expression of the target gene and effectively treating liver cancer.

[0006] Specifically, the present invention synthesizes a sulfur-containing antisense oligonucleotide YK102, whose nucleotide sequence is shown in SEQ ID NO: 1 (5'-TCCTCCGGAGCCAGACTTCA-3'), which is an oligonucleotide designed based on a tumor-related gene cell proliferation receptor IGF1R gene. The oxygen atom with a double bond on the phosphate in the YK102 oligonucleotide chain is replaced by a sulfur atom, and the molecular formula is C 192 H 245 O 99 N 72 S 19 P 19, chemical name: thymidine nucleoside-(3′→5′O,O-thiophosphate)-2′-deoxycytidine nucleoside-(3′→5′O,O-thiophosphate)-2′-deoxycytidine nucleoside-(3′→5′O,O-thiophosphate)-thymidine nucleoside-(3′→5′O,O-thiophosphate)-2′-deoxycytidine nucleoside-(3′→5′O,O-thiophosphate) Phospho)-2′-deoxycytidine-(3′→5′O,O-phosphorothioate)-2′-deoxyguanosyl-(3′→5′O,O-phosphorothioate)-2′-deoxyguanosyl-(3′→5′O,O-phosphorothioate)-2′-deoxyadenine-(3′→5′O,O-phosphorothioate)-2′-deoxyguanosyl-(3′→5′O,O-phosphorothioate) 'O,O-thiophosphate)-2'-deoxycytidine nucleoside-(3'→5'O,O-thiophosphate)-2'-deoxycytidine nucleoside-(3'→5'O,O-thiophosphate)-2'-deoxyadenine nucleoside-(3'→5'O,O-thiophosphate)-2'-deoxyguanosine nucleoside-(3'→5'O,O-thiophosphate)-2'-deoxyadenine nucleoside -(3′→5′O,O-thiophosphate)-2′-deoxycytidine-(3′→5′O,O-thiophosphate)-thymidine-(3′→5′O,O-thiophosphate)-thymidine-(3′→5′O,O-thiophosphate)-2′-deoxycytidine-(3′→5′O,O-thiophosphate)-2′-deoxyadenine.

[0007] The structural formula is:

[0008]

[0009] YK102 was synthesized using GE's OligoPilot 400 synthesizer and purified by reverse column chromatography to obtain the product.

[0010] Usually, after oral administration, thiolated oligonucleotides are quickly degraded by nucleases in the digestive tract, with little absorption and cannot be taken orally, so they are mainly administered intravenously. They are unstable under high temperature conditions and are not suitable for terminal sterilization. In order to ensure the stability of quality and the safety and effectiveness of medication, the dosage form of this product is determined to be a lyophilized powder injection.

[0011] Usually, freeze-dried preparations need to be added with freeze-dried excipients, such as mannitol, dextran and glucose. During the freezing and drying process, it can prevent the active components from sublimating and escaping with water vapor, and play a role in supporting the skeleton, so that the active components can be stably formed. For example, in the preparation method of an anti-hepatitis B immune RNA freeze-dried powder injection described in CN104940233B, dextran, mannitol, lactose monohydrate, glucose, glycine and other excipients are added; in the polypeptide freeze-dried powder injection preparation in CN103720667B, dextran, lactose and mannitol and other excipients are added; in the BCG polysaccharide nucleic acid freeze-dried powder injection in CN103405391A, excipients dextran or glucan are added.

[0012] The present disclosure unexpectedly discovered for the first time that, compared with YK102 injection preparations or lyophilized preparations to which conventional excipients, such as excipients or stabilizers such as mannitol, dextran and / or glucose, are added, YK102 lyophilized preparations without any excipients are easier to form, re-dissolve the fastest, have the best clarity, meet the quality requirements of lyophilized preparations, and have the best therapeutic effect on cancers, such as liver cancer, and the difference has reached a significant level.

[0013] In addition, compared with the YK102 freeze-dried preparations with other excipients, such as excipients or stabilizers mannitol, or dextran, the YK102 freeze-dried preparations with glucose have better formability and a reconstitution time of less than 1 minute, which meets the quality requirements for freeze-dried preparations. At the same time, they are also very effective in treating cancers such as liver cancer, and the difference has reached a significant level.

[0014] The specific technical solutions involved in this disclosure are as follows:

[0015] On the one hand, the present disclosure relates to an injection preparation, which comprises an aqueous solution of a thiocyanate antisense oligonucleotide targeting an IGF1R gene, wherein the mass ratio of the oligonucleotide to water in the oligonucleotide aqueous solution is 1:12.5-1:75, preferably 1:37.5, wherein the sequence of the thiocyanate antisense oligonucleotide targeting the IGF1R gene comprises a nucleotide sequence having more than 80% nucleotide identity with the sequence shown in SEQ ID NO:1, preferably a nucleotide sequence having more than 85%, 90%, 95%, 96%, 97%, 98%, 99% nucleotide identity; more preferably, the sequence of the thiocyanate antisense oligonucleotide targeting the IGF1R gene is as shown in SEQ ID NO:1.

[0016] In a preferred embodiment, in the injection preparation disclosed herein, the phosphorothioate antisense oligonucleotide targeting the IGF1R gene further has other chemical modifications.

[0017] In a preferred embodiment, in the injection preparation of the present disclosure, the other chemical modifications are selected from one or more of the following: locked nucleic acid modification, 2-methoxyethyl modification and 2-oxymethyl modification.

[0018] In a preferred embodiment, the injection preparation of the present disclosure further comprises an injectable pharmaceutically acceptable carrier.

[0019] In a preferred embodiment, in the injection preparation of the present disclosure, the carrier is selected from amino acids, polysaccharides, sugar alcohols or glycosides.

[0020] In a preferred embodiment, in the injection preparation of the present disclosure, the amino acid is selected from sodium glutamate, alanine, glycine, preferably sodium glutamate, wherein the mass ratio of the oligonucleotide to the amino acid is 1:0.2-1:5, preferably 1:1.

[0021] In a preferred embodiment, in the injection preparation of the present disclosure, the polysaccharide is selected from the group consisting of glucose, trehalose, and sucrose, preferably glucose, wherein the mass ratio of the oligonucleotide to the polysaccharide is 1:0.2-1:5, preferably 1:0.5.

[0022] In a preferred embodiment, in the injection preparation of the present disclosure, the polysaccharide is glucose, wherein the mass ratio of the oligonucleotide to glucose is 1:0.5 or 1:1.

[0023] In a preferred embodiment, in the injection preparation of the present disclosure, the sugar alcohol is mannitol, and the concentration is 1:0.2-1:5, preferably 1:0.5.

[0024] In a preferred embodiment, in the injection preparation of the present disclosure, the anhydroglucose is dextran, and the concentration is 1:0.2-1:5, preferably 1:0.5.

[0025] In a preferred embodiment, the injection preparation disclosed herein is contained in a container, and the container comprises a rubber stopper, and the rubber stopper is a film-coated halogenated butyl rubber stopper for sterile powder.

[0026] In a preferred embodiment, in the injection preparation of the present disclosure, the container is a sterile glass container; preferably, the sterile glass container is a neutral borosilicate glass container; the container cover is a medical container cover, preferably an aluminum-plastic composite cover.

[0027] In a preferred embodiment, in the injection preparation of the present disclosure, the container is selected from a bottle and a dish.

[0028] On the other hand, the present disclosure also relates to a method for preparing the injection preparation, which comprises the following steps:

[0029] (1) preparing an aqueous solution containing a phosphorothioate antisense oligonucleotide targeting the IGF1R gene; and

[0030] (2) Optionally, an injectable pharmaceutically acceptable carrier is added.

[0031] On the other hand, the present disclosure also relates to a lyophilized preparation, which is prepared from an aqueous solution by a step including lyophilization, wherein the aqueous solution is an aqueous solution containing a thiolated antisense oligonucleotide targeting the IGF1R gene, wherein the mass ratio of the oligonucleotide to water in the oligonucleotide aqueous solution is 1:12.5-1:75, preferably 1:37.5, wherein the sequence of the thiolated antisense oligonucleotide targeting the IGF1R gene comprises a nucleotide sequence having more than 80% nucleotide identity with the sequence shown in SEQ ID NO:1, preferably a nucleotide sequence having more than 85%, 90%, 95%, 96%, 97%, 98%, 99% nucleotide identity; more preferably, the sequence of the thiolated antisense oligonucleotide targeting the IGF1R gene is as shown in SEQ ID NO:1.

[0032] In a preferred embodiment, in the lyophilized preparation of the present disclosure, the phosphorothioate antisense oligonucleotide targeting the IGF1R gene further has other chemical modifications.

[0033] In a preferred embodiment, in the lyophilized preparation of the present disclosure, the other chemical modifications are selected from one or more of the following: locked nucleic acid modification, 2-methoxyethyl modification and 2-oxymethyl modification.

[0034] In a preferred embodiment, the lyophilized formulation of the present disclosure further comprises a lyophilizable pharmaceutically acceptable carrier.

[0035] In a preferred embodiment, in the lyophilized preparation of the present disclosure, the carrier is selected from amino acids, polysaccharides, sugar alcohols or glycosides.

[0036] In a preferred embodiment, in the lyophilized preparation of the present invention, the amino acid is selected from: sodium glutamate, alanine, glycine, preferably sodium glutamate, wherein the mass ratio of the oligonucleotide to the amino acid is 1:0.2-1:5, preferably 1:1.

[0037] In a preferred embodiment, in the lyophilized preparation of the present disclosure, the polysaccharide is selected from glucose, trehalose, sucrose, preferably glucose, wherein the mass ratio of the oligonucleotide to the polysaccharide is 1:0.2-1:5, preferably 1:0.5.

[0038] In another preferred embodiment, in the lyophilized preparation of the present disclosure, the polysaccharide is glucose, wherein the mass ratio of the oligonucleotide to glucose is 1:0.5 or 1:1.

[0039] In another preferred embodiment, in the lyophilized preparation of the present disclosure, the sugar alcohol is mannitol, and the concentration is 1:0.2-1:5, preferably 1:0.5.

[0040] In another preferred embodiment, in the lyophilized preparation of the present disclosure, the sugar anhydride is dextran, and the concentration is 1:0.2-1:5, preferably 1:0.5.

[0041] In another preferred embodiment, the lyophilized preparation of the present disclosure is contained in a container, and the container comprises a rubber stopper, and the rubber stopper is a film-coated halogenated butyl rubber stopper for sterile powder.

[0042] In another preferred embodiment, in the freeze-dried preparation of the present disclosure, the container is a sterile glass container; preferably, the sterile glass container is a neutral borosilicate glass container; and the container cover is a medical container cover, preferably an aluminum-plastic composite cover.

[0043] In another preferred embodiment, in the lyophilized preparation of the present disclosure, the container is selected from a bottle and a dish.

[0044] On the other hand, the present disclosure also relates to a method for preparing the above-mentioned lyophilized preparation, which comprises the following steps:

[0045] (1) preparing an aqueous solution comprising a phosphorothioate antisense oligonucleotide targeting the IGF1R gene; and optionally adding a lyophilizable pharmaceutically acceptable carrier to obtain a solution A; and

[0046] (2) Lyophilized solution A.

[0047] In another preferred embodiment, in the freeze-dried preparation preparation method of the present disclosure, step (2) freeze-dried solution A comprises the following steps:

[0048] a. Prefreezing: cooling to below -40°C at normal pressure, with a prefreezing time of more than 3 hours;

[0049] b. Sublimation drying: After the pre-freezing is completed, turn on the vacuum pump, raise the temperature to -20 to -10 ° C, and keep warm for 3-12 hours, preferably 4 hours;

[0050] c. Drying by sublimation: After the sublimation drying is completed, the temperature is raised to 20-40°C, preferably 40°C, and kept warm for more than 4 hours.

[0051] In another preferred embodiment, in the freeze-dried preparation preparation method of the present invention, after step c, it also includes using a screw-type plugging device in the freeze dryer to fully press the rubber plug to seal and roll the cap.

[0052] On the other hand, the present disclosure also relates to use of the injection preparation or freeze-dried preparation in preparing a drug, wherein the drug is used for treating cancer associated with the IGF1R gene.

[0053] In a preferred embodiment, in the use of the injection preparation or lyophilized preparation of the present disclosure, the cancer is selected from liver cancer, kidney cancer, stomach cancer, lung cancer, and prostate cancer.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] 1. The present invention discloses the first preparation of a sulfur-containing antisense oligonucleotide preparation, which has simple ingredients, is extremely easy to shape, has the fastest resolubility, has good clarity, and can effectively treat cancers, such as liver cancer.

[0056] 2. The present disclosure unexpectedly discovered for the first time that, compared with the YK102 lyophilized preparations to which conventional excipients, such as excipients or stabilizers such as mannitol, dextran and / or glucose, were added, the YK102 lyophilized preparations without any excipients added were easier to form, re-dissolved the fastest, had the best appearance, met the quality requirements for lyophilized preparations, and had the best therapeutic effect on cancer, such as liver cancer, and the difference also reached a significant level.

[0057] In addition, compared with the YK102 freeze-dried preparations with other excipients, such as excipients or stabilizers mannitol or dextran, the YK102 freeze-dried preparations with glucose have a reconstitution time of less than 1 minute, and their appearance is also very good, meeting the quality requirements for freeze-dried preparations. At the same time, they also have very good therapeutic effects on cancers, such as liver cancer, and the difference has reached a significant level.

[0058] 3. In addition, the present invention greatly improves the stability of the product by adopting a combination of a partially coated halogenated butyl rubber plug, an aluminum-plastic composite cover, a sterile glass bottle and an injectable oligonucleotide or its salt, and greatly reduces the probability of adhesion and shrinkage of the freeze-dried product, thereby greatly reducing the scrap rate of the enterprise in the production process, greatly reducing the production cost of the enterprise, and bringing huge economic benefits.

[0059] 4. The invention has simple process, low energy consumption, low requirements on equipment, good quality and low price, and is suitable for large-scale industrial production and application.

[0060] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0061] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 The average tumor weight of each dosage group of Prescription 2, Prescription 7 and Prescription 9 in the growth inhibition experiment of human liver cancer cell HepG2 orthotopic transplanted tumor model in nude mice.

[0063] Figure 2 In the growth inhibition experiment of human liver cancer cell HepG2 nude mouse orthotopic transplant tumor model, the tumor growth inhibition rate of each dosage group of Prescription 2, Prescription 7 and Prescription 9. DETAILED DESCRIPTION

[0064] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0065] The manufacturer of the ethylene tetrafluoroethylene copolymer film-covered chlorobutyl rubber plug used in the present invention is Hubei Huaqiang Technology Co., Ltd.

[0066] The manufacturer of the medium borosilicate glass controlled injection bottles used in this disclosure is Schott Xinkang Pharmaceutical Packaging Co., Ltd.

[0067] Example 1: Preparation of YK102

[0068] YK102, the nucleotide sequence is: 5'-TCCTCCGGAGCCAGACTTCA-3', and the structure is shown in the structural formula in the background of the invention.

[0069] The preparation method is as follows:

[0070] The instruments and reagents used are: OligoPilot 400 synthesizer from GE, USA, whose solid phase carrier is cross-linked polystyrene beads, which are connected to the initial nucleotides whose base is a modified nucleoside monomer A and whose C5′-hydroxyl group is protected by DMT;

[0071] Prepare four nucleotide solutions using acetonitrile:

[0072] monomer:

[0073] 1) DMT-dA(bz) phosphoramidite monomer:

[0074]

[0075] 2) DMT-dC(bz) phosphoramidite monomer:

[0076]

[0077] 3) DMT-dG(ibu) phosphoramidite monomer:

[0078]

[0079] 4) DMT-dT phosphoramidite monomer:

[0080]

[0081] Prepared by the following steps:

[0082] (1) Deprotection

[0083] A toluene solution of dichloroacetic acid was used as a deprotection agent to remove the DMT protecting group, followed by washing with acetonitrile.

[0084] (2) Coupling

[0085] Each nucleotide monomer was coupled in acetonitrile using 5-ethylthiotetrazolyl as an activating agent, followed by washing with acetonitrile.

[0086] (3) Thio

[0087] The thiolation was carried out using hydrogenated xanthan gum as a thiolation agent, followed by washing with acetonitrile.

[0088] (4) Hydroxyl protection

[0089] The hydroxyl group was protected using acetic anhydride as a hydroxyl group protecting agent, and then washed with acetonitrile.

[0090] The reaction is carried out in cycles according to the set sequence to obtain a fully protected product.

[0091] (5) Using dichloroacetic acid as a deprotection agent, the DMT protecting group of the last nucleotide was removed, and then acetonitrile was used for washing to obtain YK102 connected to the carrier.

[0092] (6) Aminolysis and purification

[0093] The solid phase carrier was transferred to a reactor, concentrated ammonia (25-28%) was added, and the system was cooled to room temperature after maintaining the ammonolysis at 60°C for 13 hours, and the mixture was transferred to a filter press, and eluted with a mixed solution of purified water and ethanol. The filtrate was combined, chromatographed by a reverse phase column, and freeze-dried to obtain the product, with a content of 93.8%.

[0094] Example 2: Preparation of YK102 injection

[0095] In order to investigate the molding effect of freeze-dried preparations, we selected the commonly used excipients for freeze-dried powder injections: mannitol, dextran and glucose, and designed nine prescriptions according to orthogonal experiments, plus three prescriptions in which YK102 was directly dissolved in different volumes of injection water, for a total of twelve prescriptions. Each prescription contained 20 mg of YK102.

[0096] Preparation method:

[0097] After washing with injection water, the controlled injection bottles should be free of visible foreign matter and cracks, air-dried, sterilized in an electric sterilization oven at 180°C for 2.5 hours, cooled to room temperature, and placed in the filling room for use.

[0098] Remove the outer packaging of the bag, place the butyl rubber stopper and the aluminum-plastic combination cover of the antibiotic bottle in a steam sterilizer, sterilize at 121℃±5℃ for 20 minutes, let it cool to room temperature, and put it into the filling room for use.

[0099] 1. Prescription 1

[0100] Take a certain amount of YK102 API, prepare it according to 20 mg per vial, reconstitute it with 0.5 mL of water for injection, shake it well, and let it stand. Quantitative test of the reconstituted solution.

[0101] According to the quantitative results of the reconstituted solution, dilute the solution to a concentration of 40 mg / mL with an appropriate amount of water for injection under sterile conditions and mix well; the solution is sterilized by filtering with a 0.22 μm microporous filter membrane; the filtered sample is divided into controlled injection bottles with a filling volume of 0.5 mL; and half-stopper is pressed.

[0102] Process:

[0103] Prefreezing: The samples are placed in the prefreezing room for more than 3 hours, and the indoor material temperature drops to below -40°C.

[0104] Sublimation drying: the sample was transferred to a freeze-drying chamber and treated at -20°C for 2 h; or at -10°C for 2 h.

[0105] Desorption drying: 2h at 0℃; 2h at 5℃; 4h at 10℃; 4h at 15℃; 4h at 20℃; 4h at 25℃; 4h at 30℃; 4h at 35℃; 4h at 40℃. When the product curve and vacuum curve are consistent, freeze drying is completed. Finally, the screw-type plugging device in the freeze dryer is used to fully press the rubber plug to seal, cap, fully inspect, package and put into storage.

[0106] 2. Prescription 2

[0107] According to the method in prescription 1, based on the quantitative results of the reconstituted solution, dilute the solution to a concentration of 26.67 mg / mL with an appropriate amount of water for injection under sterile conditions and mix well; the solution is sterilized by filtering with a 0.22 μm microporous filter membrane; the filtered sample is divided into controlled injection bottles with a filling volume of 0.75 mL; half-stopper is pressed.

[0108] The remaining steps are the same as Recipe 1.

[0109] 3. Prescription 3

[0110] According to the method in prescription 1, based on the quantitative results of the reconstituted solution, dilute the solution to a concentration of 20 mg / mL with an appropriate amount of water for injection under sterile conditions and mix well; the solution is sterilized by filtering with a 0.22 μm microporous filter membrane; the filtered sample is divided into controlled injection bottles with a filling volume of 1.0 mL; half-stopper is pressed.

[0111] The remaining steps are the same as Recipe 1.

[0112] 4. Prescription 4

[0113] According to the method in prescription 1, based on the quantitative results of the reconstituted solution, dilute the YK102 concentration to 40 mg / mL with an appropriate amount of 20 mg / mL mannitol injection aqueous solution under sterile conditions and mix well; the solution is sterilized by filtering with a 0.22 μm microporous filter membrane; the filtered sample is divided into controlled injection bottles with a filling volume of 0.5 mL; half-stopper is pressed.

[0114] The remaining steps are the same as Recipe 1.

[0115] 5. Prescription 5

[0116] According to the method in prescription 1, based on the quantitative results of the reconstituted solution, dilute the solution to a YK102 concentration of 40 mg / mL with an appropriate amount of 40 mg / mL dextran injection aqueous solution under sterile conditions and mix well; the solution is sterilized by filtering with a 0.22 μm microporous filter membrane; the filtered sample is divided into controlled injection bottles with a filling volume of 0.5 mL; half-stopper is pressed.

[0117] The remaining steps are the same as Recipe 1.

[0118] 6. Prescription 6

[0119] According to the method in prescription 1, based on the quantitative results of the reconstituted solution, dilute the YK102 concentration to 40 mg / mL with an appropriate amount of 60 mg / mL glucose injection aqueous solution under sterile conditions and mix well; the solution is sterilized by filtering with a 0.22 μm microporous filter membrane; the filtered sample is divided into controlled injection bottles with a filling volume of 0.5 mL; half-stopper is pressed.

[0120] The remaining steps are the same as Recipe 1.

[0121] 7. Prescription 7

[0122] According to the method in prescription 1, based on the quantitative results of the reconstituted solution, dilute the solution to a YK102 concentration of 26.67 mg / mL with an appropriate amount of 26.67 mg / mL mannitol injection aqueous solution under sterile conditions and mix well; the solution is sterilized by filtering with a 0.22 μm microporous filter membrane; the filtered sample is divided into controlled injection bottles with a filling volume of 0.75 mL; half-stopper is pressed.

[0123] The remaining steps are the same as Recipe 1.

[0124] 8. Prescription 8

[0125] According to the method in prescription 1, based on the quantitative results of the reconstituted solution, dilute the solution to a YK102 concentration of 26.67 mg / mL with an appropriate amount of 40 mg / mL dextran injection aqueous solution under sterile conditions and mix well; the solution is sterilized by filtering with a 0.22 μm microporous filter membrane; the filtered sample is divided into controlled injection bottles with a filling volume of 0.75 mL; half-stopper is pressed.

[0126] The remaining steps are the same as Recipe 1.

[0127] 9. Prescription 9

[0128] According to the method in prescription 1, based on the quantitative results of the reconstituted solution, dilute the solution to a YK102 concentration of 26.67 mg / mL with an appropriate amount of 13.33 mg / mL glucose aqueous solution for injection under sterile conditions and mix well; the solution is sterilized by filtering with a 0.22 μm microporous filter membrane; the filtered sample is divided into controlled injection bottles with a filling volume of 0.75 mL; half-stopper is pressed.

[0129] The remaining steps are the same as Recipe 1.

[0130] 10. Prescription 10

[0131] According to the method in prescription 1, based on the quantitative results of the reconstituted solution, dilute the solution to a YK102 concentration of 20 mg / mL with an appropriate amount of 30 mg / mL mannitol injection aqueous solution under sterile conditions and mix well; the solution is sterilized by filtering with a 0.22 μm microporous filter membrane; the filtered sample is divided into controlled injection bottles with a filling volume of 1.0 mL; and half-stopper is pressed.

[0132] The remaining steps are the same as Recipe 1.

[0133] 11. Prescription 11

[0134] According to the method in prescription 1, based on the quantitative results of the reconstituted solution, dilute the solution with an appropriate amount of 10 mg / mL dextran injection aqueous solution to a YK102 concentration of 20 mg / mL under sterile conditions and mix well; the solution is sterilized by filtering with a 0.22 μm microporous filter membrane; the filtered sample is divided into controlled injection bottles with a filling volume of 1.0 mL; half-stopper is pressed.

[0135] The remaining steps are the same as Recipe 1.

[0136] 12. Prescription 12

[0137] According to the method in prescription 1, based on the quantitative results of the reconstituted solution, dilute the YK102 concentration to 20 mg / mL with an appropriate amount of 20 mg / mL glucose injection aqueous solution under sterile conditions and mix well; the solution is sterilized by filtering with a 0.22 μm microporous filter membrane; the filtered sample is divided into controlled injection bottles with a filling volume of 1.0 mL; half-stopper is pressed.

[0138] The remaining steps are the same as Recipe 1.

[0139] The reconstitution time, solution pH, appearance and clarity of each formulation after freeze-drying were compared. The results are shown in Table 1.

[0140]

[0141] As shown in Table 1, the pH values ​​of the 12 formulations were similar, all ranging from 7.00 to 7.25. All formulations were clear, indicating that different formulations had no effect on the pH and clarity of the formulations.

[0142] Reconstitution time is an important quality parameter of freeze-dried powder injection. Short reconstitution time indicates good reconstitution property. After adding water, the drug can be quickly dissolved and restore the original properties of the drug solution. As can be seen from Table 1, the reconstitution time of prescription 2 is the shortest, which can be dissolved in only 43 seconds; the reconstitution time of prescriptions 4, 5, 7, 8, 10 and 11 is the longest, all greater than 10 minutes, and the reconstitution property is very poor, which does not meet the quality requirements of general freeze-dried powder injection preparations. These prescriptions all added excipients mannitol or dextran, and it can be seen that adding any of these two excipients will greatly prolong the reconstitution time; Prescriptions 6, 9 and 12 added excipient glucose, and the reconstitution time was increased by 15s, 7s and 10s respectively compared with Prescription 2, but compared with the prescriptions with the addition of mannitol or dextran, the reconstitution time was greatly shortened. It can be seen that the addition of excipients will increase the reconstitution time, but different excipients, such as the mannitol, dextran and glucose added in this embodiment, have very different effects on the reconstitution time. The reconstitution time of the preparations with the addition of mannitol or dextran is more than 10 times that of the preparations with the addition of glucose. Although no excipients were added to Prescription 1 and Prescription 3, the amount of water added was different from that in Prescription 2, that is, the concentration of YK102 before lyophilization was different from that in Prescription 2. Therefore, the reconstitution time was increased by about 35% and 33% respectively compared with Prescription 2. Therefore, it can be seen that even if no excipients were added, the different amount of water added had a great impact on the reconstitution time of the YK102 lyophilized powder injection preparation.

[0143] Different prescriptions also have a great influence on the appearance of YK102 freeze-dried powder injection: Prescription 2 is a white freeze-dried block, loose and fine, and well-formed, Prescription 1 has cracks, and Prescription 3 is shrunk. It can be seen that the different amounts of water added have a great influence on the appearance of the freeze-dried product; Prescriptions 5, 8 and 11 are all shrunk, and Prescription 8 also has a bottle spray phenomenon. Dextran is added to these three prescriptions; Prescriptions 6, 9 and 12 are slightly shrunk, and glucose is added to these three prescriptions; Prescriptions 4, 7 and 10 are well-formed, and mannitol is added to these three prescriptions. It can be seen that for YK102, not all excipients can make the product well-formed, and the addition of different excipients has a great impact on the appearance.

[0144] Through comprehensive comparison of the above 12 prescriptions, prescription 2, i.e. no excipients are added and the injection water is added to 0.75 mL, the lyophilized preparation obtained has the shortest reconstitution time, loose and delicate appearance, easy molding, good clarity, and meets the quality requirements of lyophilized preparations, and is the optimal prescription; the reconstitution time of prescriptions 1, 3, 6, 9 and 12 are also less than 1 min, with good appearance and properties, and can also be used to prepare YK102 lyophilized preparations; the reconstitution time of other prescriptions is greater than 10 min, and they cannot be used to prepare YK102 lyophilized preparations.

[0145] It can be seen that, compared with the YK102 freeze-dried preparations added with conventional excipients, such as excipients or stabilizers mannitol, dextran and / or glucose, the YK102 freeze-dried preparations without any excipients are easier to form, the fastest to re-dissolve, the difference has reached a significant level, and the appearance is the best, which meets the quality requirements of freeze-dried preparations. In addition, compared with the YK102 freeze-dried preparations added with other excipients, such as excipients or stabilizers mannitol or dextran, the YK102 freeze-dried preparations added with glucose have a re-dissolution time of within 1 minute, the difference has also reached a significant level, the appearance is also very good, and also meets the quality requirements of freeze-dried preparations.

[0146] Example 3: Growth inhibition experiment of human liver cancer cell HepG2 nude mouse orthotopic transplant tumor model

[0147] 1. Experimental Materials

[0148] Test samples: YK102 formulations 2, 7 and 9

[0149] Drug solvent: sodium chloride injection

[0150] Positive control: Sorafenib (20 mg / kg)

[0151] Experimental animals: 80 female Nu / Nu nude mice aged 4 to 6 weeks, weighing 18.0 to 20.0 g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. The test animals were kept in sterile, independently ventilated IVC cages, with 5 to 6 mice per cage. They were fed with sterilized feed specially formulated for mice and had free access to purified water. The temperature in the animal laboratory was maintained at around 25°C, the relative humidity was maintained at 40 to 70%, and the light was on for 12 hours per day.

[0152] 2. Experimental Methods

[0153] 1) Cell culture

[0154] Human hepatoma cell HepG2 was cultured in DMEM cell culture medium containing 10% fetal bovine serum (supplemented with 100uL / mL of penicillin and streptomycin), placed in a cell culture incubator at 37°C and 5% CO2, and the medium was changed every 1-2 days. Digested and passaged with 0.25% trypsin, centrifuged at 1000r / min for 5 minutes, the supernatant was discarded, and fresh culture medium was added for passage culture.

[0155] 2) Subcutaneous transplant tumor model preservation

[0156] The subcultured tumor cells were digested into a suspension under sterile conditions, rinsed with sodium chloride injection and resuspended into a suspension, which was then subcutaneously inoculated into the axilla of the right forelimb of nude mice for seed preservation.

[0157] 3) Establishment of orthotopic liver tumor model

[0158] When the subcutaneous tumor of the nude mice to be preserved grows to a diameter of about 1 to 2 cm (volume > 1000 mm 3 ), remove the tumor mass under sterile conditions, cut the tumor mass into tumor masses of about 1.0×1.0mm in size for later use. After the nude mice to be surgically inoculated are anesthetized with 0.5% pentobarbital 10mL / kg, the nude mice are fixed on the operating table, the abdominal skin is disinfected, and an incision of about 1cm is cut in the right upper abdomen to expose the liver, and the surgical hole is covered with a drape. The prepared tumor mass is placed in a special inoculation trocar, and the tumor mass is implanted into the liver with the trocar. The bleeding site of the wound is treated with sterile gauze for hemostasis. Then the liver after surgery is put back into the mouse abdominal cavity, and the abdominal muscle and skin are sutured in sequence with a 4 / 0 surgical suture needle.

[0159] 4) Experimental groups and treatment plans

[0160] The experiment set up a blank model control group, a positive control sorafenib (20 mg / kg) group, a prescription 2 low-dose (3.75 mg / kg) group, a prescription 2 medium-dose (7.5 mg / kg) group, a prescription 2 high-dose (15 mg / kg) group, a prescription 7 low-dose (3.75 mg / kg) group, a prescription 7 medium-dose (7.5 mg / kg) group and a prescription 7 high-dose (15 mg / kg) group, a prescription 9 low-dose (3.75 mg / kg) group, a prescription 9 medium-dose (7.5 mg / kg) group and a prescription 9 high-dose (15 mg / kg) group.

[0161] Three weeks after surgery, the growth of the model tumor was detected by B-ultrasound, and animals with in situ tumors and similar tumor sizes were selected and randomly divided into groups according to animal weight. The drug was injected into the tail vein once every 48 hours for a total of ten times.

[0162] After the experiment, the animals were killed by cervical dislocation. The liver was dissected and the visible tumor was removed and weighed. All dissected tissues were stored in 4% formaldehyde solution for routine pathological examination.

[0163] 5) Data processing

[0164] Weight data are expressed as X±S (mean ± standard deviation);

[0165] Tumor inhibition rate = (tumor weight of control group - tumor weight of drug-treated group) / tumor weight of control group × 100%;

[0166] The t-test program in EXCEL software was used for statistical analysis between groups and the P value was calculated. P < 0.05 was considered statistically significant.

[0167] 3. Experimental results

[0168] Table 2 Effect of YK102 preparation on the growth inhibition of human liver cancer cell HepG2 in situ nude mouse tumor model

[0169]

[0170]

[0171] 1.po. means oral administration; 2.iv. means intravenous injection

[0172] As shown in Table 2, no animal died in each group during the drug administration period. The blank model control group was compared with each drug administration group, P < 0.05, and the difference was statistically significant. Compared with the blank model control group, each drug administration group had an inhibitory effect on tumor growth, and the tumor weight was significantly reduced (see Figure 1). The tumor inhibition rate of the medium-dose (7.5 mg / kg) group of Prescription 2 was 74.4%, which was comparable to the inhibitory effect of the positive control sorafenib (20 mg / kg) group on in situ tumor growth. The tumor inhibition rate of the high-dose (15 mg / kg) group of Prescription 2 was 80.0%, which was higher than the positive control group. The tumor inhibition rates of each dose group of Prescription 7 were lower than those of each dose group of Prescription 2, among which the low-dose group was 5.6% lower than the low-dose group of Prescription 2, the medium-dose group was 8.8% lower than the medium-dose group of Prescription 2, and the high-dose group was 7.9% lower than the high-dose group of Prescription 2. The tumor inhibition rates of each dose group of Prescription 9 were 3.2%, 5.5% and 3.4% lower than those of each dose group of Prescription 2, respectively, and were 2.4%, 3.3% and 4.5% higher than those of each dose group of Prescription 7, respectively (see Figure 2 ). This indicates that different formulations and processes have very different effects in vivo, and formulations 2 and 9 have stronger tumor inhibition effects in animals than formulation 7.

[0173] Compared with the YK102 freeze-dried preparations added with conventional excipients, such as excipients or stabilizers mannitol, dextran and / or glucose, the YK102 freeze-dried preparations without any excipients have the best therapeutic effect on cancer, such as liver cancer, and the difference has reached a significant level. In addition, compared with the YK102 freeze-dried preparations added with other excipients, such as excipients or stabilizers mannitol or dextran, the YK102 freeze-dried preparations added with glucose also have a very good therapeutic effect on cancer, such as liver cancer, and the difference has also reached a significant level.

[0174] Although the present disclosure has been described in detail above with general descriptions and specific implementation schemes, it is obvious to those skilled in the art that some modifications or improvements may be made to the present disclosure. Therefore, these modifications or improvements made without departing from the spirit of the present disclosure are within the scope of protection claimed by the present disclosure.

Claims

1. A lyophilized preparation, which is prepared from an aqueous solution by a freeze-drying step, wherein the aqueous solution is an aqueous solution of a thiocyanate antisense oligonucleotide targeting an IGF1R gene, in which the mass ratio of the oligonucleotide to water is 1:37.5, and the sequence of the thiocyanate antisense oligonucleotide targeting an IGF1R gene is shown in SEQ ID NO: 1, in, The lyophilized preparation is prepared by a method comprising the following steps: (1) preparing an aqueous solution of a phosphorothioate antisense oligonucleotide targeting the IGF1R gene to obtain a solution A; and (2) Lyophilized solution A; The step (2) of freeze-drying solution A comprises the following steps: a. Pre-freezing: cooling to below -40℃ under normal pressure, the pre-freezing time is more than 3h; b. Sublimation drying: After pre-freezing, the sample is transferred to the freeze-drying chamber and treated at -20℃ for 2h; or at -10℃ for 2h; c. Desorption drying: treat at 0℃ for 2h; treat at 5℃ for 2h; treat at 10℃ for 4h; treat at 15℃ for 4h; treat at 20℃ for 4h; treat at 25℃ for 4h; treat at 30℃ for 4h; treat at 35℃ for 4h; treat at 40℃ for 4h.

2. A method for preparing the lyophilized preparation according to claim 1, comprising the following steps: (1) preparing an aqueous solution of a phosphorothioate antisense oligonucleotide targeting the IGF1R gene to obtain a solution A; and (2) Lyophilized solution A; Wherein, the freeze-dried solution A in step (2) comprises the following steps: a. Pre-freezing: cooling to below -40℃ under normal pressure, the pre-freezing time is more than 3h; b. Sublimation drying: After pre-freezing, the sample is transferred to the freeze-drying chamber and treated at -20℃ for 2h; or at -10℃ for 2h; c. Desorption drying: treat at 0℃ for 2h; treat at 5℃ for 2h; treat at 10℃ for 4h; treat at 15℃ for 4h; treat at 20℃ for 4h; treat at 25℃ for 4h; treat at 30℃ for 4h; treat at 35℃ for 4h; treat at 40℃ for 4h.

3. The method according to claim 2 further comprises, after step c, using a screw-type plugging device in a freeze dryer to fully press the rubber plug to seal and roll the cap.

4. Use of the lyophilized preparation of claim 1 in the preparation of a medicament for treating cancer associated with the IGF1R gene, wherein the cancer is selected from the group consisting of liver cancer and kidney cancer.

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