An F-base albumin drug delivery system, its preparation method and use

Abstract

The present invention discloses an F-base albumin drug delivery system, its preparation method and uses, including Step 1: introducing a trifluoromethyl base analogue (F-base) into a nucleotide chain; Step 2: linking the trifluoromethyl base analogue (F-base) described in Step 1 to a drug. Due to the adoption of the above method, an F-base albumin drug delivery system and its pharmaceutical composition are constructed for targeted drug delivery to tumors, ultimately achieving the purpose of enhancing the binding of nucleic acid to plasma proteins, thereby protecting it from degradation by ribozymes, improving its extremely easy renal clearance, prolonging its circulation time in vivo, enhancing its enrichment at the target site, and promoting the internalization of the drug in tumor cells and the exertion of the anti-tumor effect.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid drug targeted delivery, and particularly to an F-base albumin drug delivery system and its preparation method and use. Background Art

[0002] Nucleoside drugs

[0003] Nucleosides and deoxynucleosides are the basic components of ribonucleic acid and deoxyribonucleic acid. As derivatives of natural nucleosides, nucleoside drugs can block the synthesis of DNA or RNA and disrupt the balance of the natural nucleoside pool, thereby achieving the purpose of blocking virus replication or cell proliferation. Therefore, they are widely used in the treatment of viral infectious diseases, tumors, etc. Among them, 50% of the drugs for antiviral treatment are nucleoside drugs, such as lamivudine for anti-hepatitis virus, cidofovir for anti-herpes virus, ganciclovir for anti-cytomegalovirus, stavudine for anti-retroviral virus, etc. Antitumor drugs such as 5-fluorouracil, cytarabine, gemcitabine, etc. also belong to nucleoside drugs.

[0004] Targeted delivery of nucleoside drugs

[0005] Nucleoside drugs are limited by nucleoside transport receptors on the cell surface and deoxycytidine kinase and thymidine kinase inside the cell. Therefore, the targeted delivery of nucleoside drugs is crucial. Currently, it mainly relies on hydrogels, liposomes, nanomaterials, fatty chain modification, nucleic acid aptamer modification, etc. However, there are some problems with the current targeted delivery. For example, 5-FU drugs are prone to leakage from liposomes, and nucleic acids modified with fatty chains accelerate the clearance of drugs due to their liver targeting, resulting in problems such as short in vivo circulation time and large drug dosage.

[0006] Small molecule drugs

[0007] Small molecule drugs such as MMAE often need to be linked with targeted molecules such as antibodies due to their high toxicity to reduce toxicity. However, antibodies have problems such as too long in vivo circulation time, uncontrollable drug loading, batch-to-batch differences, etc., and currently there is no protein that is only expressed in tumors, so tumor site specificity cannot be fully achieved.

[0008] Therefore, there is a need for a drug delivery system and its preparation method in the field of tumor drug targeted delivery that can extend the in vivo circulation time of drugs, reduce the dosage and frequency of drugs, and have good specificity.

[0009] Trifluoromethyl modification

[0010] Trifluoromethyl is an artificial base that can be modified at the end of nucleic acids through solid-phase synthesis to increase the stability of nucleic acids. Because the action of trifluoromethyl can promote the binding of nucleic acids to albumin, thereby prolonging the circulation time of nucleic acids in the body. Because its binding to membrane proteins increases, nucleoside drugs are more easily internalized into cells to exert their effects. Summary of the Invention

[0011] In order to better solve at least one of the above problems, the present invention provides a method for preparing an F-base albumin drug delivery system.

[0012] The above method includes:

[0013] Step 1: Introduce a trifluoromethyl base analogue (F-base) into the nucleotide chain;

[0014] Step 2: Connect the trifluoromethyl base analogue (F-base) described in Step 1 to the drug.

[0015] In the above method, the connection method is solid-phase synthesis.

[0016] In the above method, the drug is selected from one or a combination of 5-FU, gemcitabine (Gem), and MMAE.

[0017] In the above method, the trifluoromethyl base analogue is located in the middle and / or at the end of the nucleotide chain.

[0018] In the above method, the nucleotide sequence modified by the trifluoromethyl base analogue is shown as SEQ ID No.: 1-3:

[0019] SEQ1: FF-5FU-5FU-5FU-5FU-5FU-5FU-FF;

[0020] SEQ2: FF-Gem-Gem-Gem-Gem-FF;

[0021] SEQ3: FF-TTTT-FF-MMAE.

[0022] In the above method, the 5-FU and gemcitabine (Gem) are 5FU nucleic acid sequences and gemcitabine (Gem) nucleic acid sequences synthesized by solid-phase synthesis, and the MMAE is the MMAE sequence linked to the nucleic acid sequence; the conditions for linking the MMAE sequence are: Vc-MMAE reacts with a thiol-modified DNA / RNA sequence overnight in a solution containing acetonitrile and 400 mM KCl.

[0023] In the above method, Step 1 further includes:

[0024] Step 1.1: Synthesis. RNA and primers are synthesized on a synthesizer.

[0025] Step 1.2: Deprotection by decolorization. After synthesis, deprotection by decolorization is carried out in an AMA solution at 65 °C for 30 min.

[0026] Step 1.3: Purification. Purification is carried out using a C-18 column by reverse-phase high-performance liquid chromatography.

[0027] Step 1.4: Elution. Elution is carried out using 0.1 M triethylammonium acetate (TEAA) buffer and acetonitrile as eluents.

[0028] On the other hand, the present invention also provides an F-base albumin drug delivery system prepared by the above method. In the above system, a drug composition and a nucleotide sequence modified with a trifluoromethyl base analogue are included.

[0029] On the other hand, the present invention also provides the use of the above drug composition for targeted drug delivery to tumors.

[0030] The advantages and beneficial effects of the present invention are as follows:

[0031] ① Introduce a trifluoromethyl base analogue at the end or middle position of the nucleic acid to enhance the binding of the drug to plasma proteins and protect it from degradation by ribozymes.

[0032] ② Improve the circulation time of the drug in vivo through the interaction between the trifluoromethyl base analogue and albumin.

[0033] ③ Enhance the enrichment of the drug at the target site through the interaction between the trifluoromethyl base analogue and albumin, increase the drug content at the tumor site, and at the same time reduce the drug dosage and toxic and side effects.

[0034] ④ The present patent utilizes the interaction between the F-base and albumin to achieve the targeted delivery of various drugs to the tumor site, demonstrating the universality of the F-base albumin drug delivery system. Description of the Drawings

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0036] Figure 1 It is a fluorescence signal intensity change diagram reflecting the enrichment of the F-base modified 5-FU sequence at the tumor site in the embodiments of the present invention.

[0037] Figure 2It is a graph showing the change in tumor size after treatment with the 5-FU sequence modified with F base in the embodiments of the present invention;

[0038] Figure 3 It is a graph showing the change in fluorescence signal intensity reflecting the enrichment of the gemcitabine sequence modified with F base in the tumor site in the embodiments of the present invention.

[0039] Figure 4 It is a graph showing the change in tumor size after treatment with the gemcitabine sequence modified with F base in the embodiments of the present invention;

[0040] Figure 5 It is a graph showing the change in fluorescence signal intensity reflecting the enrichment of the MMAE sequence modified with F base in the tumor site in the embodiments of the present invention;

[0041] Figure 6 It is a broken line graph showing the change in tumor size after treatment with the MMAE sequence modified with F base in the embodiments of the present invention. Detailed implementation manners

[0042] The following combines the accompanying drawings and embodiments to further describe the detailed implementation manners of the present invention. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention and cannot be used to limit the protection scope of the present invention.

[0043] The present invention provides an F-base albumin drug delivery system, its preparation method and uses, and also relates to a pharmaceutical composition containing the F-base albumin drug delivery system. One or several embodiments will be introduced below.

[0044] Step 1: Introduce a trifluoromethyl base analogue (F base) into the nucleotide chain. Preferably, introduce the trifluoromethyl base analogue (F base) in the middle or at the end of the nucleotide chain, or introduce it at both the middle and the end simultaneously;

[0045] Step 1.1: Synthesis. RNA and primers are synthesized on a synthesizer;

[0046] Step 1.2: Deprotection by decolorization. After synthesis, deprotect by decolorization in an AMA solution at 65°C for 30 min;

[0047] Step 1.3: Purification. Use a C-18 column and purify by reverse-phase high performance liquid chromatography;

[0048] Step 1.4: Elution. Use 0.1 M triethylammonium acetate (TEAA) buffer and acetonitrile as eluents for elution.

[0049] Step 2: Connect the trifluoromethyl base analogue (F base) described in Step 1 to the drug by solid-phase synthesis.

[0050] As a preferred embodiment, the drug is selected from one or a combination of 5-FU, gemcitabine (Gem), and MMAE. Through solid-phase synthesis, this patent exemplarily constructs a series of sequences containing 5-FU, gemcitabine (Gem), and MMAE, and modifies the trifluoromethyl group at the end or middle position of the nucleic acid (SEQ ID No.: 1-3), as shown in Table 1 (SEQ ID No.: 1-3 in the table does not have a specific nucleotide (DNA) sequence, but only exemplarily shows the positions of F base, 5-FU, gemcitabine (Gem), and MMAE in the nucleotide sequence):

[0051]

[0052] Table 1: Example diagram of nucleotide sequences modified with trifluoromethyl

[0053] Among them, 5-FU and gemcitabine (Gem) are 5FU nucleic acid sequences and gemcitabine (Gem) nucleic acid sequences synthesized by solid-phase synthesis, and MMAE is the MMAE sequence linked to the nucleic acid sequence; among them, the condition for linking the MMAE sequence is: Vc-MMAE reacts overnight with the DNA / RNA sequence modified with thiol in a solution containing acetonitrile and 400 mM KCl. Preferably, 100 ul of the above KCl solution and 150 ul of acetonitrile are used.

[0054] In the following Examples 1-3, the stability and efficacy of the above example sequences in tumor treatment will be experimentally tested.

[0055] Example 1

[0056] In vivo targeting test of F-modified 5-FU sequence

[0057] The in vivo targeting of the F-modified 5-FU sequence was investigated through in vivo experiments on mice. The Cy5-modified 5-FU sequence (10 uM, 100 ul) was injected into tumor-bearing mice (MIA-paca2 cells (pancreatic cancer cells)) via the tail vein, and the fluorescence changes at the tumor site in the mice were observed within 0-48 hours. As Figure 1 shown, the results showed that the F-modified 5-FU sequence continuously accumulated at the tumor site, and there was still a strong fluorescence signal at the tumor site after the fluorescence in the kidneys disappeared at 48 hours.

[0058] Efficacy test of F-modified 5-FU sequence

[0059] The in vivo efficacy of the F-modified 5-FU sequence was investigated through in vivo experiments on mice. The 5-FU sequence or the F-modified 5FU sequence was injected into tumor-bearing mice (MIA-paca2 cells (pancreatic cancer cells)) via the tail vein (5 mg / kg, 3 times a day), and the size changes of the tumors in the mice were observed. As Figure 2As shown, the results show that the treatment effect of F-modified 5-FU is better.

[0060] Example 2

[0061] In vivo targeting test of F-modified gemcitabine sequence

[0062] The in vivo stability of the F-modified gemcitabine sequence was investigated through in vivo experiments in mice. The Cy5-modified sequence (10 uM, 100 ul) was injected into a pancreatic cancer mouse model through the tail vein, and the fluorescence changes at the tumor site in the mice were observed within 0 - 48 hours. As Figure 3 shown, the results show that the F-modified gemcitabine sequence continuously accumulates at the tumor site, and there is still a strong fluorescence signal at the tumor site after the fluorescence in the kidneys disappears at 48 hours.

[0063] Efficacy test of F-modified gemcitabine sequence

[0064] The in vivo efficacy of the F-modified gemcitabine sequence was investigated through in vivo experiments in mice. The gemcitabine sequence or the F-modified gemcitabine sequence was injected into a pancreatic cancer mouse model through the tail vein (5 mg / kg, 3 times a day), and the size changes of the tumors in the mice were observed. As Figure 4 shown, the results show that the treatment effect of the F-modified gemcitabine sequence is better.

[0065] Example 3

[0066] In vivo targeting test of F-modified MMAE sequence

[0067] The in vivo stability of the F-modified MMAE sequence was investigated through in vivo experiments in mice. The Cy5-modified sequence (10 uM, 100 ul) was injected into a pancreatic cancer mouse model through the tail vein, and the fluorescence changes at the tumor site in the mice were observed within 0 - 48 hours. As Figure 5 shown, the results show that the F-modified MMAE sequence continuously accumulates at the tumor site, and there is still a strong fluorescence signal at the tumor site after the fluorescence in the kidneys disappears at 48 hours.

[0068] Efficacy test of F-modified MMAE sequence

[0069] The in vivo efficacy of the F-modified gemcitabine sequence was investigated through in vivo experiments in mice. The gemcitabine sequence or the F-modified MMAE sequence was injected into a pancreatic cancer mouse model through the tail vein (5 mg / kg, 3 times a day), and the size changes of the tumors in the mice were observed. As Figure 6 shown, the results show that the treatment effect of the F-modified MMAE sequence is better.

[0070] The preparation method of the F-base albumin drug delivery system described in the present invention can enhance the binding of nucleic acids to plasma proteins, thereby protecting them from degradation by ribozymes. At the in vivo level, nucleic acids modified with trifluoromethyl or its analogs can be protected from cleavage and their rapid renal clearance can be improved due to their binding to plasma proteins, prolonging their circulation time in the body and enhancing their enrichment at the target site.

[0071] Through the above method, the present invention can prepare relevant F-base albumin drug delivery systems. Drugs or drug combinations such as 5-FU, gemcitabine, MMAE, etc. are made into nucleic acid chains by solid-phase synthesis, and trifluoromethyl base analogs are modified at the end or middle position of the nucleic acid by solid-phase synthesis, optimizing the position and number of modifications.

[0072] Meanwhile, by using the drug combination containing the F-base albumin drug delivery system described in the present invention, the increased binding of nucleic acids to membrane proteins due to the F-base at the tumor site can promote the internalization of drugs in tumor cells and the exertion of anti-tumor effects, enabling the nucleic acid drug combination to not only maintain its original toxicity but also bind to plasma proteins, realizing the targeted drug delivery for tumors and completing the targeted delivery at the tumor site.

[0073] The above description of the disclosed embodiments is provided so that those skilled in the art of the present technology can implement or use the present application. Various modifications to these embodiments will be obvious to those skilled in the art of the present technology. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but rather will conform to the widest scope consistent with the principles and novel features disclosed herein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of an F-base albumin drug delivery system, characterized in that Comprising: Step 1: Construct a nucleic acid strand containing a drug; Step 2: Modify the middle and / or end of the nucleic acid strand containing the drug described in Step 1 with trifluoromethyl by solid-phase synthesis to form a trifluoromethyl-modified drug sequence; The nucleic acid strand containing the drug includes a 5FU nucleic acid sequence, a gemcitabine (Gem) nucleic acid sequence, and an MMAE sequence linked to the nucleic acid sequence; The 5FU nucleic acid sequence and the gemcitabine (Gem) nucleic acid sequence are synthesized by solid-phase synthesis of 5-FU and gemcitabine (Gem). The condition for linking the MMAE sequence is that Vc-MMAE reacts overnight with a DNA / RNA sequence modified with a thiol group in a solution containing acetonitrile and 400 mM KCl; The drug sequences include three types as shown in SEQ ID No.: 1-3: SEQ ID No.1: FF-5FU-5FU-5FU-5FU-5FU-5FU-FF; SEQ ID No.2: FF-Gem-Gem-Gem-Gem-FF; SEQ ID No.3: FF-TTTT-FF-MMAE.

2. A pharmaceutical composition, characterized in that, Comprising the trifluoromethyl-modified drug sequence obtained by the preparation method described in Claim 1.

3. Use of a pharmaceutical composition as claimed in claim 2, characterized in that, Preparation of a drug for tumor-targeted therapy.

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