WO2023160354A1 - 新型酸敏感性适配体雷公藤甲素偶联物及应用 - Google Patents

新型酸敏感性适配体雷公藤甲素偶联物及应用 Download PDF

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WO2023160354A1
WO2023160354A1 PCT/CN2023/074352 CN2023074352W WO2023160354A1 WO 2023160354 A1 WO2023160354 A1 WO 2023160354A1 CN 2023074352 W CN2023074352 W CN 2023074352W WO 2023160354 A1 WO2023160354 A1 WO 2023160354A1
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triptolide
aptamer
conjugate
tumor
acid
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French (fr)
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鲁军
邓赟
陈瑶
杨济瑞
左艺
黎潇
任青
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成都中医药大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to the field of medicine and chemical industry, and mainly relates to a triptolide conjugate of aptamer sensitive to weak acid microenvironment of tumors; in particular, it relates to a novel acid-sensitive aptamer conjugate of triptolide and its application.
  • Triptolide also known as triptolide and triptolide alcohol, is an epoxy diterpene lactone compound extracted from the root, leaf, flower and fruit of Euonymus plant Tripterygium wilfordii.
  • Alkaloids such as tergenine, tripterine, tripterine, tripterine and tripterine constitute the main active ingredients of tripterygium extract, which are insoluble in water but easily soluble in methanol, dimethyl Sulfoxide, absolute ethanol, ethyl acetate, chloroform, etc.
  • Triptolide has broad-spectrum antitumor activity, can inhibit the proliferation of various tumor cells, and induce apoptosis and autophagy.
  • triptolide The antitumor activity of triptolide is better than traditional anticancer drugs such as adriamycin and paclitaxel, and it can effectively inhibit the proliferation of tumor cells at very low concentrations (2-10ng/mL).
  • triptolide can also fight against tumor drug resistance, increase the sensitivity of tumor cells to other antitumor drugs, and play a synergistic effect in combination with chemotherapy drugs and ionizing radiation.
  • MUC1 mucin 1
  • MDA-MB-231 negative breast cancer MDA-MB-231
  • lung cancer A549 lung cancer A549
  • colon cancer HCT116 colon cancer HCT116
  • Nucleic acid aptamer is a single-stranded oligonucleotide that uses its self-folding advanced structure to bind to a specific target, mainly referring to a piece of DNA (deoxyribonucleic acid), RNA (ribonucleic acid) or modified DNA and RNA .
  • Nucleic acid aptamers can bind to target molecules through 3D conformational complementarity, have similar affinity and specificity to antibodies, and have the advantages of good water solubility, low immunogenicity, easy production, low cost and good stability, and Clinical trials have proven to be safe. Due to the ability to achieve specific delivery at the cellular level, nucleic acid aptamers have been widely used as targeting molecules in the study of targeted drug delivery.
  • the aptamer AS1411 can specifically bind to the highly expressed nucleolin on the surface of tumor cells, and the nucleolin on the membrane surface can promote the large pinocytosis of AS1411 by tumor cells function, increase the uptake, and AS1411 can form a G-quadruplex structure to enhance its stability; the aptamer E07 can specifically bind to the epidermal growth factor receptor EGFR.
  • Apt, AS1411, and E07 aptamers all have the characteristics of good water solubility, strong stability and high protein expression in tumor cells, and can be used as carriers for drug delivery.
  • triptolide As a broad-spectrum anticancer drug, triptolide has a strong antitumor effect, but it also has large toxic and side effects and poor water solubility. Therefore, the modified triptolide is coupled with the aptamer, so as to use the targeting function of the aptamer to transport triptolide to a specific tumor location, and bind to the highly expressed receptor on the cell membrane, making triptolide A hormone targets and kills tumors while avoiding the killing effect on normal cells.
  • the ethylenyl ether bond is used as an acidic link to connect triptolide and the aptamer.
  • the ethylenic ether bond can only be broken when the pH is below 4. This results in a lower release efficiency of the aptamer triptolide conjugate formed through the linkage in tumor cells, and a poorer inhibitory effect on cancer cells.
  • the purpose of the present invention is to overcome the low acid sensitivity of the linker in the aptamer triptolide conjugate existing in the prior art, which is not easy to break in the environment of cancer cells in the body, resulting in insufficient triptolide directional release , and thus cause the defect that the inhibitory effect of cancer cells is not obvious, a new linker is designed as the coupling arm between triptolide and the aptamer, specifically, a new acid-sensitive aptamer triptolide is provided A protein conjugate and its application.
  • A is a nucleic acid aptamer
  • B is a link connecting triptolide and the nucleic acid aptamer, and the link forms a functional group of an acetal ester at the 14th hydroxyl position of triptolide.
  • the conjugate delivered triptolide to tumor cells and endocytosed into lysosomes; based on the characteristics of the acidic environment of lysosomes, acetal linkage The key releases the complete triptolide in the acidic environment of the lysosome to kill tumor cells.
  • A is a nucleic acid aptamer
  • B is the link connecting triptolide and the nucleic acid aptamer, and the link has an acetal functional group at the 14th hydroxyl position of triptolide.
  • the conjugate is two triptolides coupled to one nucleic acid aptamer.
  • the general structure of the connecting key B is as follows:
  • triptolide found that its 14th hydroxyl group is the active site and at the same time it is a modification site, and the linkage B is connected with the 14th hydroxyl group of triptolide to form an acetal linkage.
  • the key can block the toxic effect of triptolide on normal tissues, and at the same time release the complete triptolide in the acidic environment of lysosomes to achieve the precise treatment of cancer.
  • the encircled position is a new chemical bond formed between the connecting bond B and the 14th hydroxyl group of triptolide, which is defined as the acetal bond for this R bond.
  • Cleavage mechanism in an acidic environment, hydrogen ions attack the oxygen atom (1) at the end of the ester group in the acetal ester bond of the aptamer triptolide conjugate, forming an unstable metasalt intermediate (2), leading to further
  • the cleavage of form contains aptamer linkage part (3) and extremely unstable triptolide intermediate (4) containing carbocation, and then a molecule of water combines with the carbocation of triptolide intermediate ( 5), the carbocation proton is transferred to the oxygen atom in water (6), and then the proton is transferred to the oxygen atom of triptolide, forming an unstable triptolide hemiacetal structure (7), the hemiacetal
  • the carbon-oxygen bond in the aldehyde structure is broken to form triptolide and formaldehyde.
  • 0 ⁇ n 1 ⁇ 100 preferably, 0 ⁇ n 1 ⁇ 50, more preferably, 0 ⁇ n 1 ⁇ 20;
  • 0 ⁇ n 2 ⁇ 100 preferably, 0 ⁇ n 2 ⁇ 50, more preferably, 0 ⁇ n 2 ⁇ 20;
  • n 1 and n 2 includes 0, but not 0 at the same time
  • n 1 and n 2 are preferably 2, 3, 5, or 10.
  • the nucleic acid aptamer is a modified nucleic acid aptamer, and the general structural formula of the modified nucleic acid aptamer is A 1 -C 1 or C 1 -A 1 -C 1 Among them, the structure of C1 is:
  • 0 ⁇ n 3 ⁇ 100 preferably, 0 ⁇ n 3 ⁇ 50, more preferably, 0 ⁇ n 3 ⁇ 20;
  • 0 ⁇ n 4 ⁇ 100 preferably, 0 ⁇ n 4 ⁇ 50, more preferably, 0 ⁇ n 4 ⁇ 20;
  • n 3 and n 4 includes 0, but not 0 at the same time
  • n 3 and n 4 are preferably 2, 3, 5, or 10.
  • the conjugate is obtained by reacting a triptolide derivative with a nucleic acid aptamer modification.
  • the triptolide derivatives are obtained by reacting the 14-position hydroxyl group of triptolide with the link B in an organic solvent, and the 14-position hydroxyl group of triptolide forms a triptolide containing an acetal functional group. A derivatives.
  • the conjugate is obtained by any one of substitution reaction, cyclization reaction or addition reaction of the B2 functional group on the triptolide derivative. Specifically, a substitution reaction between an amino group and a carboxyl group; or an addition reaction between a mercapto group and a maleamide group, or a cyclization reaction between an azido group and an alkynyl group.
  • the nucleic acid aptamer includes any one of AS1411, Pegaptanib, Sgc8c, A10, DNAaptamer, RNAaptamer, CL4, Apt and E07.
  • the sequence of the nucleic acid aptamer is as follows:
  • Pegaptanib GCGAACCGAUGGAAUUUUUGGACGCUCGC;
  • Sgc8c ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA;
  • A10 GGGAGGACGAUGCGGAUCAGCCAUGUUUACGUCACUCCUUGUCAAUCCCUCAUCGGCAGACGACUCGCCCGA;
  • DNA aptamer GGGAGACAAGAATAAACGCTCAA-(25N)-TTCGACAGGAGGCTCACAACAGGC;
  • RNA aptamer GGGGGCAUACUUGUGAGACUUUUAUGUCACCCCC;
  • a method for preparing a triptolide conjugate according to the acid-sensitive aptamer which specifically comprises the following steps:
  • Step 1 preparing triptolide derivatives, in which triptolide has an acetal functional group attached to the 14th hydroxyl position of triptolide;
  • Step 2 modifying the nucleic acid aptamer to obtain the modified nucleic acid aptamer modification A 1 -C 1 ;
  • Step 3 linking the triptolide derivative and the nucleic acid aptamer modification in step 2 through the B2 linkage to obtain the acid-sensitive aptamer triptolide conjugate.
  • the acid-sensitive rametine aptamer conjugate can be prepared by those skilled in the art in combination with the contents of the present invention and attempts in the field;
  • acetal ester functional group on the link between triptolide and the aptamer, that is, to connect the 14th hydroxyl group of triptolide to the aptamer
  • Linkages between ligands form linkages with acetal functionality.
  • the aptamer blocks the active group of triptolide, resulting in the incomplete release of triptolide. Compared with simple triptolide As far as the aptamer is concerned, the cytotoxicity of the conjugate is very low, which indicates that the aptamer will not damage normal cells in the human body after entering the human body. It is less toxic to the human body.
  • the aptamer triptolide conjugate provided by the present invention has good tumor targeting in vivo, and at the same time avoids other tissues to achieve the effect of tumor targeting; intravenous injection experiments show that AS1411-TP has good tumor targeting in vivo It has good anti-tumor effect, and the connection key used to connect triptolide and aptamer has played an obvious role in vivo.
  • the conjugate does not break in normal tissue and has low toxicity; but breaks in tumor tissue to release triptolide and play a therapeutic role.
  • Fig. 1 is the MS spectrum of compound 5;
  • Fig. 2 is the MS spectrum of compound 7;
  • Fig. 3 is the MS spectrum of compound 9;
  • Fig. 4 is the HPLC analysis graph of the stability of the conjugate (AS1411-TP) at different time points in serum;
  • Fig. 5 is the HPLC analysis chart of the stability of the conjugate (E07-TP) at different time points in serum;
  • Fig. 6 is the HPLC analysis chart of the release of triptolide in different pH values of the conjugate (AS1411-TP);
  • Fig. 7 is the HPLC analysis chart of the release situation of triptolide in different pH values of the conjugate (E07-TP);
  • Fig. 8 is the HPLC analysis chart of the release of triptolide in different pH values of the conjugate (the AS1411-TP conjugate linked by an ene ether bond);
  • Fig. 9 is the chemical formula of AS1411-TP-1;
  • Figure 10 is the inhibitory effect curve of AS1411-TP, TP, AS1411 on the colon cancer cell line HCT116;
  • Figure 11 is a graph showing the inhibitory effect of AS1411-TP, TP, and AS1411 on the lung adenocarcinoma cell line A549;
  • Figure 12 is a graph showing the inhibitory effect of AS1411-TP, TP, and AS1411 on the breast cancer cell line MDA-MB-231;
  • Figure 13 is a graph showing the inhibitory effect of AS1411-TP, TP, and AS1411 on the pancreatic cancer cell line PANC-1;
  • Figure 14 is a graph showing the inhibitory effect of AS1411-TP, TP, and AS1411 on the liver cancer cell line HepG2;
  • Figure 15 is a graph showing the toxic effects of AS1411-TP, TP, and AS1411 on human embryonic kidney cells HEK293;
  • Figure 16 is a curve of LO2 toxicity of AS1411-TP, TP, and AS1411 to normal human liver cells;
  • Figure 17 shows the distribution of AS1411-TP, TP, and AS1411 in different tissues of the tumor-bearing mouse colon cancer model
  • Figure 18 shows the distribution of AS1411-TP, TP, and AS1411 in different tissues of the tumor-bearing mouse breast cancer model
  • Figure 19 shows the distribution of AS1411-TP, TP, and AS1411 in different tissues of the tumor-bearing mouse lung cancer model
  • Figure 20 is a diagram of AS1411-TP, TP, AS1411 anti-mice colon cancer xenograft tumor
  • Figure 21 is a diagram of AS1411-TP, TP, AS1411 anti-mice breast cancer xenograft tumors
  • Figure 22 is a diagram of AS1411-TP, TP, AS1411 anti-mouse lung cancer xenograft tumors
  • Figure 23 is an analysis chart of the in vivo stability test data of AS1411-TP, TP, and AS1411;
  • This example provides an acid-sensitive aptamer triptolide conjugate labeled Apt-TP.
  • the structural formula and reaction route of the conjugate are as follows:
  • Aptamer 1 is an amino-modified Apt synthesized by the nucleic acid synthesizer AKTAOligopilot100system; specifically, aptamer 1: NH 2 -(CH2) 6 -5'GCAGTTGATCCTTTGGATACCCTGG3'MS:7843;
  • This example provides an acid-sensitive aptamer triptolide conjugate, labeled as E07-TP.
  • the structural formula and reaction route of the conjugate are as follows:
  • Aptamer 2 is thiol PEG-modified E07 synthesized by the nucleic acid synthesizer AKTAOligopilot100system;
  • aptamer 2 Specifically, aptamer 2:
  • the preparation process of conjugate 7 comprises the following steps:
  • This example provides an acid-sensitive aptamer triptolide conjugate labeled AS1411-TP.
  • the structural formula and reaction route of the conjugate are as follows:
  • Aptamer 3 is azide PEG-modified AS1411 synthesized by the nucleic acid synthesizer AKTAOligopilot100system; aptamer 3 is
  • the preparation process of conjugate 7 comprises the following steps:
  • AS1411-TP conjugate (linked by acetal ester bond), E07-TP conjugate (linked by acetal ester bond), AS1411-TP-1 conjugate (linked by ether bond), mouse serum was obtained from BALB/c mice, ⁇ , 7-8 weeks old (Beijing Speifu Biotechnology Co., Ltd.)
  • the tumor microenvironment is a weakly acidic environment, and the sensitivity of the conjugate to the weakly acidic microenvironment determines the therapeutic effect of the drug.
  • AS1411-TP-1 conjugate (linked by ether bond), AS1411-TP conjugate (linked by acetal ester bond), E07-TP conjugate (linked by acetal ester bond) and dissolve it in 100 ⁇ L of PBS buffer solution was used to simulate the tumor microenvironment, and HPLC was used to measure the content changes of the conjugates under different pH conditions after 2 hours.
  • AS1411-TP conjugate acetal ester bond connection
  • AS1411-TP-1 the control group
  • AS1411-TP-1 chemical formula of AS1411-TP-1 is shown in Figure 9;
  • Figure 6 is the HPLC assay chart of AS1411-TP conjugate
  • Figure 7 is the HPLC assay chart of E07-TP conjugate
  • Figure 8 is AS1411-TP-1
  • the nucleolin aptamer AS1411 is a highly water-soluble single-stranded oligonucleotide that can specifically bind to the highly expressed nucleolin protein on the surface of tumor cell membranes. Therefore, the nucleolin aptamer- Taking triptolide conjugates as an example, research on in vitro activity, in vitro toxicity, in vivo distribution, in vivo activity, in vivo stability, etc., to fully characterize and verify the performance of the linking bonds in the conjugate of the present invention and Viability of suppressing cancer cells in vivo.
  • Triptolide (TP) was provided by Shanghai Yaji Biotechnology Co., Ltd.; the aptamer (AS1411) was obtained from Chengdu Hittor Pharmaceutical Development Co., Ltd.; trypsin, thiazolyl blue (MTT) (Biofroxx, Germany); dimethyl Sulfoxide (DMSO) (MPBiomedicals, France); fetal bovine serum (FBS) (Newzerum, New Zealand); DMEM medium, RPMI1640 medium, culture dish, 96-well plate (Corning, USA); colon cancer cells HCT116, Breast cancer cells MDA-MB-231, non-small cell lung cancer cells A549, pancreatic cancer cells PANC-1, and liver cancer cells HepG2 were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences and preserved in our laboratory.
  • the tumor cell lines HCT116, MDA-MB-231, A549, PANC-1, HepG2 were taken out from the liquid nitrogen tank and placed in 37 In a water bath at °C, after the frozen cells are thawed into a cell suspension, centrifuge at a speed of 1000r/min for 3min, discard the supernatant, and add 1ml of fresh complete medium containing 10% fetal bovine serum and 1% double antibody Mix the cells evenly, then add 5ml of medium, and gently blow the cells repeatedly with a pipette gun to make them evenly mixed, and finally transfer the cell suspension to a 10mm culture dish, and mark the cell name, cell generation number, recovery date and The name of the operator, placed in the cell culture incubator for cultivation.
  • HCT116, PANC-1, and HepG2 cell lines were cultured in DMEM medium
  • MDA-MB-231 cell lines and A549 cell lines were cultured in RPMI1640 medium).
  • HCT116, MDA-MB-231, A549, PANC-1, HepG2 cells in good growth state remove the medium, add 1ml sterile PBS to wash, add 1.5ml trypsin to digest the cells, the cells become round and fall off after about 2min, Add 1.5ml of medium to stop digestion, transfer to a centrifuge tube, centrifuge at 1000r/min for 3min, discard the supernatant, add 1ml of fresh medium to resuspend, then add fresh medium for 1:3 ratio to other culture dishes, Mark the subculture date and generation number, and place them in a cell culture incubator for culture.
  • HCT116, MDA-MB-231, A549, PANC-1, and HepG2 cells in good growth state were taken, and the same cell passage experiment was performed. After the cells were resuspended, the cells were counted with a hemocytometer, and 2500 cells/well were planted in 96 In the orifice plate, after the cells were cultured for 24 hours, the original medium was removed, and the medium containing different concentrations of drugs was added to continue the culture for 48 hours.
  • Table 1 is the data summary table of the cell proliferation inhibition rate after AS1411-TP, TP, AS1411 acted on the colon cancer cell line HCT116 for 48h
  • Table 2 is the data summary table of the cell proliferation inhibition rate after AS1411-TP, TP, AS1411 acted on the lung adenocarcinoma cell line A549 for 48h
  • Table 3 is the data summary table of the cell proliferation inhibition rate after AS1411-TP, TP, AS1411 acted on the breast cancer cell line MDA-MB-231 for 48h
  • Table 4 is the data summary table of the cell proliferation inhibition rate after AS1411-TP, TP, AS1411 acted on the pancreatic cancer cell line PANC-1 for 48h
  • Table 5 shows the cell proliferation inhibition rate of AS1411-TP, TP, and AS1411 after acting on the liver cancer cell line HepG2 for 48h Data summary table for
  • triptolide has a significant effect on colon cancer cells HCT116, breast cancer cells MDA-MB-231
  • the IC 50 of lung adenocarcinoma cell A549, pancreatic cancer cell PANC-1 and liver cancer cell HepG2 were 18.88 ⁇ 0.13nM, 22.38 ⁇ 0.24nM, 30.23 ⁇ 0.17nM, 14.4 ⁇ 0.09nM, 10.46 ⁇ 0.33nM;
  • the IC 50 for colon cancer cell HCT116, breast cancer cell MDA-MB-231, lung adenocarcinoma cell A549, pancreatic cancer cell PANC-1, and liver cancer cell HepG2 are 20.01 ⁇ 0.12nM, 20.03 ⁇ 0.27nM, 29.87 ⁇ 0.16nM, respectively , 11.18 ⁇ 0.03nM, 11.21 ⁇ 0.26nM.
  • AS1411 had no obvious inhibitory effect on any tumor cells.
  • AS1411-TP has an anti-tumor effect equivalent to that of triptolide on various tumor cells and is not the effect of AS1411, indicating that the connection of aptamers does not affect the anti-tumor effect of triptolide, which also proves that the tumor Microenvironment-responsive acid-sensitive linkages can be broken within tumor cells to release triptolide.
  • human normal liver cells LO2 and human embryonic kidney cells HEK293 were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences and preserved by our laboratory.
  • LO2 and HEK293 cell lines Take out the tumor cells LO2 and HEK293 cell lines from the liquid nitrogen tank, place them in a 37°C water bath, and after the frozen cells melt into a cell suspension, centrifuge at 1000r/min for 3min, discard the supernatant, and add 1ml Fresh complete medium containing 10% fetal bovine serum and 1% double antibody to mix the cells evenly, then add 5ml of medium, gently blow the cells repeatedly with a pipette gun to make them evenly mixed, and finally transfer the cell suspension to In a 10mm culture dish, mark the cell name, cell generation number, recovery date and the name of the operator, and place it in a cell culture incubator. (HEK293 cell line is cultured in DMEM medium, LO2 cell line is cultured in RPMI1640 medium)
  • Table 6 shows the cell proliferation inhibition rate data after AS1411-TP, TP, AS1411 acted on human embryonic kidney cells HEK293 for 48h
  • Table 7 shows the cell proliferation inhibition rate data of AS1411-TP, TP, AS1411 after 48 hours of LO2 action on normal human liver cells
  • Triptolide has greater cytotoxicity to normal liver cell LO2 and normal human embryonic kidney cell HEK293, with IC 50 of 9.01 ⁇ 0.25nM and 5.98 ⁇ 0.21nM, respectively.
  • IC 50 of the conjugate against LO2 and HEK293 are 51.86 ⁇ 0.13nM, 48.10 ⁇ 0.13nM, respectively, AS1411 against the two Normal cells had no obvious inhibitory effect. This fully proves that the connection of the aptamer reduces the cytotoxicity of triptolide to normal cells.
  • the reason may exist in the following two aspects: 1.
  • the aptamer blocks the active group of triptolide, and normal cells are in a non-weakly acidic environment, so triptolide cannot be completely released.
  • the expression of nucleolin on the cell membrane surface of normal liver cells and kidney cells is less, the AS1411 aptamer has no targeting effect on normal cells, the cells have less intake of the conjugate, and the toxic effect is small.
  • the dose of AS1411-TP administration group was set at 50 ⁇ M, and the administration volume was 100 ⁇ L.
  • the dose of triptolide (TP) administration group was set at 50 ⁇ M, and the administration volume was 100 ⁇ L.
  • the same materials as in the second part above will not be repeated here, except for the experimental animals: BALB/cnu mice, ⁇ , 6-7 weeks old. Provided by SPEF (Beijing) Biotechnology Co., Ltd.; kept in the experimental animal room of Chengdu University of Traditional Chinese Medicine.
  • mice Feed female nude mice for 6-7 weeks under standard conditions, suspend colon cancer cell HCT116 in serum-free medium, and inoculate each mouse with 3*10 6 tumor cells.
  • the mice were randomly divided into two groups, and AS1411-TP and TP were injected into the tail vein respectively, with 12 mice in each group.
  • the animals were sacrificed 8 hours after administration, and the hearts, livers, spleens, lungs, kidneys and tumor tissues were taken out, rinsed with normal saline, weighed, and frozen for a short time at -80°C for later use.
  • the distribution results in Figure 17 show that AS1411-TP is more distributed in colon cancer tumor tissues, less in normal tissues, and more TP is distributed in normal liver and kidney tissues.
  • the content of AS1411-TP in normal mouse tissues is much lower than that of TP, while in tumor tissues, the percentage of AS1411-TP experimental group to the total injected amount in tumor tissue is higher than that of triptolide experimental group, indicating that AS1411 -TP has good tumor tissue targeting of colon cancer in vivo.
  • mice Feed female nude mice for 6-7 weeks under standard conditions, suspend breast cancer cells MDA-MB-231 in serum-free medium, and inoculate 7*10 6 tumor cells per mouse.
  • the mice were randomly divided into two groups, and the aptamer-triptolide conjugate and triptolide were injected into the tail vein respectively, with 12 mice in each group.
  • the animals were sacrificed 4 hours after administration, and the hearts, livers, spleens, lungs, kidneys and tumor tissues were taken out, rinsed with normal saline, weighed, and frozen at -80°C for a short time for later use.
  • the content of the aptamer-triptolide and triptolide in the tissue was detected by HPLC. Take out the organs and tissues, add 2 times the amount of normal saline to the tissue, homogenize, and centrifuge to filter, take 90 ⁇ L of filtrate, add 10 ⁇ L of standard solution to the filtrate, and use HPLC to determine the drug content in each tissue after administration at different times after filtration.
  • HPLC HPLC
  • AS1411-TP is more distributed in breast cancer tumor tissues, less in normal tissues, and triptolide is more distributed in normal liver and kidney tissues.
  • the content of AS1411-TP in normal mouse tissues was much lower than that of triptolide; while in tumor tissues, the amount of AS1411-TP in the experimental group accounted for the total injected amount The percentage is higher than that of the triptolide experimental group, indicating that AS1411-TP has better breast cancer tumor tissue targeting in vivo.
  • mice Feed female nude mice for 6-7 weeks under a standard environment, suspend lung cancer cell A549 in serum-free medium, and inoculate 6*10 6 tumor cells per mouse.
  • the mice were randomly divided into two groups, and the aptamer-triptolide conjugate and triptolide were injected into the tail vein respectively, with 12 mice in each group.
  • the animals were sacrificed 4 hours after administration, and the hearts, livers, spleens, lungs, kidneys and tumor tissues were taken out, rinsed with normal saline, weighed, and frozen for a short time at -20°C for future use.
  • the content of AS1411-TP and TP in the tissue was detected by HPLC.
  • the contents of the aptamer-triptolide conjugate and triptolide in the tissue were detected by HPLC. Take out the organs and tissues, add 2 times the amount of normal saline to the tissue, homogenize, and centrifuge to filter, take 90 ⁇ L of filtrate, add 10 ⁇ L of standard solution to the filtrate, and use HPLC to determine the drug content in each tissue after administration at different times after filtration.
  • HPLC HPLC
  • AS1411-TP is more distributed in tumor tissues, less in normal tissues, and triptolide is more distributed in normal liver and kidney tissues.
  • the content of AS1411-TP in normal tissues of mice is much lower than that of triptolide, while in tumor tissues, the percentage of AS1411-TP experimental group in tumor tissues to the total injected amount is higher than that of triptolide experimental group , indicating that AS1411-TP has good tumor targeting of lung cancer cell A549 in vivo, and at the same time avoids other tissues to achieve the effect of tumor targeting.
  • mice Feed female nude mice for 6-7 weeks under standard environment, suspend colon cancer cell HCT116 in serum-free medium, and inoculate 5*10 6 tumor cells per mouse.
  • the mice were randomly divided into 4 groups, and the aptamer-triptolide conjugate, triptolide, and aptamer were injected into the tail vein respectively, with 12 mice in each group.
  • a PBS negative control group was set up.
  • Dosing group TP (10 ⁇ M, 30 ⁇ M and 50 ⁇ M, administration volume is 100 ⁇ L), AS1411 (10 ⁇ M, 30 ⁇ M and 50 ⁇ M, administration volume is 100 ⁇ L) and AS1411-TP (10 ⁇ M, 30 ⁇ M and 50 ⁇ M, administration volume is 100 ⁇ L) .
  • the drug was administered once every 2 days for 8 consecutive times, and the experiment was terminated 24 hours after the last administration. After the experiment, the animals were killed by breaking the cervical vertebrae, the tumors were peeled off, the tumors were weighed, and the inhibitory rate of the drugs on tumor growth was calculated. The t test was used to compare the tumor weight, tumor volume, RTV and other indicators of animals in each group.
  • Table 8 The experimental data are summarized in Table 8, and Figure 20 shows the anti-mice colon cancer xenograft tumors obtained by different injection drugs.
  • Table 8 is the summary table of the experimental data of AS1411-TP, TP, AS1411 on the growth inhibitory effect of the colon cancer tumor model in tumor-bearing mice
  • the body weight of the mice in the TP, AS1411-TP, and AS1411 groups was basically maintained within the tolerable range of animal toxicity and side effects during the 8 times of administration.
  • the body weight of the mice in the PBS negative control group showed a slow and gradual decline trend, and compared with the beginning of grouping, the average body weight decreased by 1.7g. From the animal tumor weight, tumor inhibition rate and RTV value, it can be seen that the tumor growth rate and tumor inhibition rate of mice in the TP group, AS1411-TP group, and AS1411 group were slower than those in the PBS group and showed a dose-dependent relationship.
  • AS1411-TP 10 ⁇ M, 30 ⁇ M and 50 ⁇ M were administered intravenously to colon cancer model nude mice for 8 consecutive times, and the tumor growth was significantly inhibited, and the inhibitory effect was better than that of TP administered at the same concentration, indicating that AS1411-TP was in vivo It has good antitumor effect.
  • the materials used in the experiment are the same as those tested in 5.1 colon cancer cell lines, the only difference is that the tumor strains are different;
  • mice Feed female nude mice for 6-7 weeks under a standard environment, suspend breast cancer cell line MDA-MB-231 in serum-free medium, and inoculate 6*10 6 tumor cells per mouse.
  • the mice were randomly divided into 4 groups, and the aptamer-triptolide conjugate, triptolide, and aptamer were injected into the tail vein respectively, with 12 mice in each group.
  • a PBS negative control group was set up.
  • Administration groups triptolide (10 ⁇ M, 30 ⁇ M and 50 ⁇ M), AS1411 (10 ⁇ M, 30 ⁇ M and 50 ⁇ M) and AS1411-TP (10 ⁇ M, 30 ⁇ M and 50 ⁇ M).
  • the drug was administered once every 2 days for 8 consecutive times, and the experiment was terminated 24 hours after the last administration. After the experiment, the animals were killed by breaking the cervical vertebrae, the tumors were peeled off, the tumors were weighed, and the inhibitory rate of the drugs on tumor growth was calculated. The t test was used to compare the tumor weight, tumor volume, RTV and other indicators of animals in each group.
  • the experimental data are summarized in Table 9, and Figure 21 shows the anti-mice breast cancer xenograft tumors obtained by injecting different drugs.
  • Table 9 Summary table of experimental data of AS1411-TP, TP, AS1411 on growth inhibition of tumor-bearing mouse breast cancer model
  • the body weight of the mice in the TP, AS1411-TP, and AS1411 groups was basically maintained within the tolerable range of animal toxicity and side effects during the 8 times of administration.
  • the body weight of the mice in the PBS negative control group showed a slow and gradual decline trend, and compared with the beginning of grouping, the average body weight decreased by 2.1g.
  • the tumor weight, tumor inhibition rate and RTV value of the animals, the tumor growth rate and tumor inhibition rate of mice in the TP group, AS1411-TP, and AS1411 administration groups were slower than those in the PBS group and showed a dose-dependent relationship.
  • mice Feed female nude mice for 6-7 weeks under a standard environment, suspend the lung cancer cell line A549 in serum-free medium, and inoculate 6*10 6 tumor cells per mouse.
  • the mice were randomly divided into 4 groups, and AS1411-TP, triptolide, and aptamer were injected into the tail vein respectively, with 12 mice in each group.
  • a PBS negative control group was set up.
  • the drug was administered once every 2 days for 8 consecutive times, and the experiment was terminated 24 hours after the last administration. After the experiment, the animals were killed by breaking the cervical vertebrae, the tumors were peeled off, the tumors were weighed, and the inhibitory rate of the drugs on tumor growth was calculated. The t test was used to compare the tumor weight, tumor volume, RTV and other indicators of animals in each group.
  • Table 10 The experimental data are summarized in Table 10, and Figure 22 shows the anti-mouse lung cancer xenograft tumors obtained by different injection drugs.
  • Table 10 is the summary table of the experimental data of AS1411-TP, TP, AS1411 on the growth inhibitory effect of tumor-bearing mice lung adenocarcinoma tumor model
  • the body weight of the mice in the TP, AS1411-TP, and AS1411 groups was basically maintained within the tolerable range of animal toxicity and side effects during the 8 times of administration.
  • the body weight of the mice in the PBS negative control group showed a slow and gradual decline trend, and compared with the beginning of grouping, the average body weight decreased by 2.0g. From the animal tumor weight, tumor inhibition rate, RTV value, etc., it can be seen that the tumor growth rate and tumor inhibition rate of mice in the TP group, AS1411-TP, and AS1411 administration groups were slower than those in the PBS group and showed a dose-dependent relationship.
  • the stability experiment of the aptamer triptolide conjugate in mice was carried out to simulate the stability of the drug in the human body, so as to verify the degree of dissociation and stability of the conjugate in different tissues.
  • mice 27 tumor-bearing mice were randomly selected, and 100 ⁇ L of normal saline solution with a concentration of 50 ⁇ MAS1411-TP was injected into the caudal vein.
  • 3 mice were killed at each place, anesthetized by intraperitoneal injection of pentobarbital sodium, blood was collected by heart puncture, heart, liver, spleen, and lung , kidney, solid tumor and other tissues, after tissue homogenization, extraction, sample processing and pre-experiment to determine the retention time of TP, AS1411 and AS1411-TP conjugates respectively, HPLC detects the free TP in each tissue at each time point and the amount of AS1411-TP, and calculate the release efficiency, monitor and quantitatively investigate the stability of the drug in the tissue.
  • Table 11 The experimental data are shown in Table 11, and the analysis results of the in vivo stability test data are shown in Figure 23.
  • Table 11 is the in vivo stability test data of the aptamer triptolide conjugate (AS1411-TP)
  • Figure 23 is an analysis chart of the in vivo stability test data of the aptamer triptolide conjugate (AS1411-TP).
  • AS1411-TP the aptamer triptolide conjugate
  • Table 11 and Figure 16 it can be shown that: after 36 hours, the AS1411-TP conjugate There is almost no dissociation or only a small amount of dissociation in plasma, heart, spleen, lung and other normal tissues, and slightly more dissociation in kidney and liver tissues; while in tumor tissues, AS1411-TP conjugates dissociate in large quantities, and The degree of dissociation increases over time.
  • the above results prove that the linker used to connect triptolide and the aptamer plays a significant role in vivo.
  • the conjugate does not break in normal tissue and has low toxicity; but breaks in tumor tissue to release triptolide and play a therapeutic role.
  • the aptamer triptolide conjugate provided by the present invention has an antitumor effect equivalent to that of the original drug both in vivo and in vitro, which proves that the connection of the aptamer does not affect the antitumor effect of the conjugate.
  • both in vivo and in vitro experiments showed that the toxicity of the conjugate was reduced compared with the original drug, which proved that the conjugate had a better targeting anti-tumor effect and could avoid toxicity to normal tissues.
  • the distribution of the aptamer triptolide conjugate provided by the present invention is much higher than that in other tissues, and the dissociation efficiency in tumor tissue is much higher than that in normal tissues, which proves that the conjugate provided by the present invention It has good tumor targeting in vivo, and at the same time, the tumor microenvironment can meet the release conditions of the conjugate.

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Abstract

一种肿瘤弱酸微环境敏感性适配体雷公藤甲素偶联物，雷公藤甲素的14位羟基与适配体之间通过缩醛酯连接键偶联。该连接键为酸性敏感型连接键，其断裂条件为pH=3.5-6.5，在肿瘤微环境下更容易断裂。基于适配体靶向肿瘤细胞膜表面高表达蛋白的特点，该偶联物将雷公藤甲素靶向递送至肿瘤细胞并介导内吞作用到达溶酶体内；基于溶酶体酸性环境的特点，缩醛酯连接键在溶酶体酸性环境下释放出完整的雷公藤甲素，对肿瘤细胞靶向杀伤。克服了现有技术中适配体雷公藤甲素偶联物中的连接键酸敏感性低，在体内癌细胞环境下不易断裂，雷公藤甲素定向释放不足，造成癌细胞抑制效果不明显的缺陷。

Description

新型酸敏感性适配体雷公藤甲素偶联物及应用 技术领域
本发明涉及医药化工领域，主要涉及肿瘤弱酸微环境敏感性适配体雷公藤甲素偶联物；具体涉及新型酸敏感性适配体雷公藤甲素偶联物及应用。
背景技术
雷公藤甲素又称雷公藤内酯、雷公藤内酯醇，是从卫矛科植物雷公藤的根、叶、花及果实中提取的一种环氧二萜内酯化合物，与雷公藤碱、雷公藤次碱、雷公藤晋碱、雷公藤春碱、雷公藤增碱和雷公藤明碱等生物碱构成了雷公藤提取物的主要活性成分，难溶于水，易溶于甲醇、二甲基亚砜、无水乙醇、乙酸乙酯、氯仿等。雷公藤甲素具有广谱的抗肿瘤活性，能抑制多种肿瘤细胞的增殖，并诱导细胞凋亡和自噬。雷公藤甲素抗肿瘤活性优于阿霉素、紫杉醇等传统的抗癌药物在极低浓度(2-10ng/mL)下即可有效抑制肿瘤细胞增殖。除此之外，雷公藤甲素还可以对抗肿瘤耐药，并提高肿瘤细胞对其他抗肿瘤药物的敏感性、与化疗药物和电离辐射相结合发挥协同效应。
癌症目前已经成为人们生命安全的主要威胁之一。肿瘤细胞与正常细胞的区别之一在于肿瘤细胞中某些蛋白会发生异常表达。例如黏蛋白1(MUC1)在乳腺癌细胞(MCF-7)及其耐药细胞系膜表面都异常表达，核仁素与表皮生长因子受体EGFR也是在肿瘤细胞异常表达的蛋白，前者在三阴性乳腺癌MDA-MB-231、肺癌A549以及结肠癌HCT116等细胞系中都异常表达，后者通常是作为肺癌治疗的靶点。
核酸适配体(aptamer)是一种利用自身折叠的高级结构与特定靶标结合的单链寡核苷酸，主要是指一段DNA(脱氧核糖核酸)、RNA(核糖核酸)或者修饰的DNA和RNA。核酸适配体可以通过3D构象互补与靶分子结合，具有与抗体相似的亲和性和特异性，且具有水溶性好，免疫原性低，生产容易，花费少及稳定性好等优点，并且临床实验证明是安全的。由于可以实现细胞水平的特异性递送，核酸适配体作为靶向分子已经被广泛应用在药物靶向递送研究中，例如黏蛋白1(MUC1)核酸适配体Apt能与MUC1发生特异性、高亲和力结合且其序列较短，容易合成和纯化；适配体AS1411可与肿瘤细胞表面高表达的核仁素特异性结合，同时膜表面的核仁素又能促进肿瘤细胞对AS1411的大胞饮作用，增加摄取量，而AS1411能形成G-四联体结构增强其稳定性；适配体E07能与表皮生长因子受体EGFR特异性结合。
Apt、AS1411、E07适配体都具有水溶性好，稳定性强并靶向肿瘤细胞高表达蛋白的特点，可作为药物运输的载体。雷公藤甲素作为广谱抗癌药物，其抗肿瘤作用强，但其毒副作用也大、水溶性较差。因此将修饰后的雷公藤甲素与适配体偶联，从而利用适配体的靶向功能将雷公藤甲素运到特定的肿瘤位置，与细胞膜上高表达受体结合，使其雷公藤甲素对肿瘤靶向杀伤，同时避免对正常细胞的杀伤作用。
肿瘤细胞除了特异性高表达某些蛋白外，由于肿瘤细胞需要通过浸润周围组织实现细胞的增殖和转移，所以肿瘤组织中的溶酶体含量远高于正常组织。溶酶体是真核细胞中重要的细胞降解器，维持细胞的代谢稳定，其特点之一是细胞器内为酸性环境(PH＝3.5-5.5)。
现有技术中，将烯醚键作为酸性连接键连接雷公藤甲素和适配体，研究过程中发现，烯醚键在在pH＝4以下时才可断裂。这导致通过该连接键形成的适配体雷公藤甲素偶联物在肿瘤细胞中的释放效率较低，癌细胞抑制效果较差。
发明内容
本发明的目的在于克服现有技术中所存在的适配体雷公藤甲素偶联物中的连接键酸敏感性低，在体内癌细胞环境下不易断裂，导致雷公藤甲素定向释放量不足，进而造成癌细胞抑制效果不明显的缺陷，设计一种新的连接键作为雷公藤甲素与适配体之间的偶联臂，具体的，提供一种新型酸敏感性适配体雷公藤甲素偶联物及应用。
为了实现上述发明目的，本发明提供了以下技术方案：
一种酸敏感性适配体雷公藤甲素偶联物，所述偶联物通式如下：
其中，A为核酸适配体；B为连接雷公藤甲素与核酸适配体的连接键，所述连接键在雷公藤甲素的第14位羟基位置形成有缩醛酯的官能团。
本发明技术方案公开的一种适配体-雷公藤甲素偶联物，雷公藤甲素的14位羟基与适配体之间通过缩醛与酯基形成的连接键(缩醛酯官能团)偶联，该连接键为酸性敏感型连接键，其断裂条件为(pH＝3.5-6.5)，相较于现在已有的烯醚连接键(断裂pH3.5以下)，其pH值的敏感度更低，在肿瘤微环境下更容易断裂。基于适配体靶向肿瘤细胞膜表面高表达蛋白的特点，该偶联物将雷公藤甲素靶向递送至肿瘤细胞并内吞至溶酶体内；基于溶酶体酸性环境的特点，缩醛连接键在溶酶体酸性环境下释放出完整的雷公藤甲素，对肿瘤细胞进行靶向杀伤。
一种酸敏感性适配体雷公藤甲素偶联物，所述偶联物通式如下式所示：
其中，A为核酸适配体；
B为连接雷公藤甲素与核酸适配体的连接键，所述连接键在雷公藤甲素的第14位羟基位置形成有缩醛酯官能团。该偶联物为一个核酸适配体上偶联两个雷公藤甲素。
作为本发明的优选技术方案，连接键B的通式结构如下：
对雷公藤甲素的构效关系进行研究发现，其14位羟基是活性位点，同时又是修饰位点，连接键B与雷公藤甲素第14位羟基连接形成缩醛连接键，该连接键可封闭雷公藤甲素对正常组织的毒性作用，同时在溶酶体酸性环境下释放出完整的雷公藤甲素，达到精准治疗癌症的作用。
如上式所示，圈住的位置即是连接键B与雷公藤甲素第14位羟基形成的一种新的化学键，此处定义为该种R键为缩醛酯键。
如下式所示为该酸敏感性适配体雷公藤甲素偶联物的反应
裂解机制:在酸性环境中，氢离子进攻适配体雷公藤甲素偶合物的缩醛酯键中的酯基端的氧原子(1)，形成不稳定的佯盐中间体(2)，导致进一步的断裂形成含有适配体连接键部分(3)和极不稳定的含有碳正离子的雷公藤甲素中间体(4)，然后一分子水与雷公藤甲素中间体的碳正离子结合(5)，碳正离子质子转移至水中的氧原子上(6)，然后质子再转移至雷公藤甲素的氧原子上，形成不稳定的雷公藤甲素半缩醛结构(7)，半缩醛结构中碳氧键发生断裂形成雷公藤甲素与甲醛。
进一步优选的，B1的结构通式结构如下所示：
其中，0≤n1≤100，优选的，0≤n1≤50，更优选的，0≤n1≤20；
其中，0≤n2≤100，优选的，0≤n2≤50，更优选的，0≤n2≤20；
n1和n2的取值范围包括0，但不同时为0；
具体的，n1、n2取值范围优选为2、3、5、10。
本方案中，针对B1，其中0≤n1(n2)≤100，但烷烃链或烷氧基链的调节设计可对：雷 公藤甲素-适配体与其靶蛋白的结合的干扰，适当的加大连接键与肿瘤微环境的接触面积，提高雷公藤甲素的释放速率。但更长的烷烃链合成成本高，且产率低，因此更优选的1≤n1(n2)≤20。n1和n2的取值范围包括0，但不同时为0；具体的，n1、n2取值范围优选为2、3、5、10。
作为本发明的优选技术方案，所述核酸适配体是经过修饰的核酸适配体，所述经过修饰的核酸适配体结构通式为A1-C1或C1-A1-C1其中，C1的结构为：
其中，0≤n3≤100，优选的，0≤n3≤50，更优选的，0≤n3≤20；
其中，0≤n4≤100，优选的，0≤n4≤50，更优选的，0≤n4≤20；
n3和n4的取值范围包括0，但不同时为0；
具体的，n3、n4取值范围优选为2、3、5、10。
本方案中，针对C1，其中0≤n3≤100，但烷烃链或烷氧基链的设计可雷公藤甲素对适配体与其靶蛋白的结合的干扰，同时，可加大连接键与肿瘤微环境的接触面积，增加雷公藤甲素的释放速率。但更长的烷烃链合成成本高，且产率低，因此优选的，0≤n3(n4)≤50，更优选的0≤n3(n4)≤20。n3和n4的取值范围包括0，但不同时为0；具体的，n3、n4取值范围优选为2、3、5、10。
作为本发明的优选技术方案，所述偶联物通过雷公藤甲素衍生物与核酸适配体修饰物反应得到。具体的，所述雷公藤甲素衍生物通过雷公藤甲素14位羟基在有机溶剂中与连接键B反应后得到，所述与雷公藤甲素14位羟基形成含有缩醛酯官能团的雷公藤甲素衍生物。
作为本发明的优选技术方案，所述偶联物的通式为如下结构：
所述偶联物通过雷公藤甲素衍生物上的B2官能团通过取代反应、环化反应或加成反应中的任意一种反应得到。具体的，氨基与羧基的取代反应；或巯基与马来酰胺之间的加成反应，或者叠氮基与炔基之间的环化反应。
作为本发明的优选技术方案，
B2的通式如下：
所述核酸适配体包括AS1411、Pegaptanib、Sgc8c、A10、DNAaptamer、RNAaptamer、CL4、Apt和E07中的任意一种。
核酸适配体的序列如下所示：
AS1411：GGTGGTGGTGGTTGTGGTGGTGGTGG；
Pegaptanib：GCGAACCGAUGGAAUUUUUGGACGCUCGC；
Sgc8c：ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA；
A10：GGGAGGACGAUGCGGAUCAGCCAUGUUUACGUCACUCCUUGUCAAUCCUCAUCGGCAGACGACUCGCCCGA；
DNA aptamer：GGGAGACAAGAATAAACGCTCAA-(25N)-TTCGACAGGAGGCTCACAACAGGC；
RNA aptamer：GGGGGCAUACUUGUGAGACUUUUAUGUCACCCCC；
CL4：GCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAGGC；
Apt：GCAGTTGATCCTTTGGATACCCTGG；
E07：GGACGGAUUUAAUCGCCGUAGAAAAGCAUGUCAAAGCCGGAACCGUCC。
作为本发明的优选技术方案，提供一种根据所述的酸敏感性适配体雷公藤甲素偶联物的制备方法，具体包括如下步骤制备得到：
步骤1：制备雷公藤甲素衍生物，所述雷公藤甲素衍生物中，雷公藤甲素的第14位羟基位置连接有缩醛酯官能团；
步骤2：对核酸适配体进行修饰得到经过修饰的核酸适配体修饰物A1-C1；
步骤3：将雷公藤甲素衍生物与步骤2中的核酸适配体修饰物通过B2连接键连接即得酸敏感性适配体雷公藤甲素偶联物。
优选的，所述酸敏感性雷甲素适配体偶联物可以由本领域技术人员结合本发明记载的内容及本领域尝试进行制备；
将雷公藤甲素的第14位羟基通过与连接键形成缩醛酯官能团的方式进行封闭，得到雷公藤甲素衍生物；同步的，对适配体先进行烷烃或烷氧烃链的修饰；再根据与雷公藤甲素衍生物的端基的官能团类型，再对适配体连接烷烃或烷氧烃链的一端进行官能团修饰；最后将雷公藤甲素衍生物与修饰后的适配体在催化试剂作用下，利用核酸适配体上的氨基或者巯基或者叠氮基和雷公藤甲素衍生物的羧基或者马来酰胺或者叠氮进行反应得到偶联物。两者连接后即形成上文所述的B2连接键。
一种如上所述的酸敏感性雷甲素适配体偶联物在制备抗肿瘤药物中的应用。
与现有技术相比，本发明的有益效果：
1、本发明提供的适配体雷公藤甲素偶联物中，雷公藤甲素与适配体之间的连接键上具有缩醛酯官能团，即连接雷公藤甲素第14位羟基与适配体之间的连接键形成了具有缩醛酯官能团的连接键。该连接键为酸性敏感型连接键，其断裂条件为(pH＝3.5-6.5)，pH值敏感度更低，具有较好的酸响应性，在肿瘤微环境下更容易断裂。
2、本发明提供的适配体雷公藤甲素偶联物中，以AS1411-TP进行体外活性研究表明，AS1411-TP对各肿瘤细胞均有与雷公藤甲素相当的抗肿瘤效果，适配体的连接并不影响雷公藤甲素的抗肿瘤作用，同时证明肿瘤微环境响应性酸性敏感连接键可在肿瘤细胞内断裂，释放出雷公藤甲素。
3、本发明提供的适配体雷公藤甲素偶联物在正常细胞环境中，适配体封闭了雷公藤甲素的活性基团，导致雷公藤甲素无法完整释放，相较单纯的雷公藤甲素而言，偶联物的细胞毒性很低，这表明，该适配体进入人体后，不会损伤人体内的正常的细胞。对人体的毒性较小。
4、本发明提供的适配体雷公藤甲素偶联物体内有较好的肿瘤靶向性，同时规避其他组织，达到肿瘤靶向的效果；静脉注射给药实验表明，AS1411-TP在体内具有较好的抗肿瘤作用，用于连接雷公藤甲素和适配体的连接键在体内起到了明显的作用。使得偶联物在正常组织中不发生断裂，毒性较小；而在肿瘤组织中断裂，释放出雷公藤甲素，发挥治疗作用。
附图说明
图1为化合物5的MS图谱；
图2为化合物7的MS图谱；
图3为化合物9的MS图谱；
图4为偶联物(AS1411-TP)在血清中不同时间点的稳定性的HPLC分析图；
图5为偶联物(E07-TP)在血清中不同时间点的稳定性的HPLC分析图；
图6为偶联物(AS1411-TP)在不同pH值中雷公藤甲素释放情况的HPLC分析图；
图7为偶联物(E07-TP)在不同pH值中雷公藤甲素释放情况的HPLC分析图；
图8为偶联物(以烯醚键连接的AS1411-TP偶联物)在不同pH值中雷公藤甲素释放情况的HPLC分析图；
图9为AS1411-TP-1的化学式；
图10为AS1411-TP、TP、AS1411对结肠癌细胞系HCT116的抑制作用曲线；
图11为AS1411-TP、TP、AS1411对肺腺癌细胞系A549的抑制作用曲线图；
图12为AS1411-TP、TP、AS1411对乳腺癌细胞系MDA-MB-231的抑制作用曲线图；
图13为AS1411-TP、TP、AS1411对胰腺癌细胞系PANC-1的抑制作用曲线图；
图14为AS1411-TP、TP、AS1411对肝癌细胞系HepG2的抑制作用曲线图；
图15为AS1411-TP、TP、AS1411对人胚胎肾细胞HEK293的毒性作用曲线图；
图16为AS1411-TP、TP、AS1411对人正常肝细胞LO2毒性曲线图；
图17为AS1411-TP、TP、AS1411在荷瘤小鼠结肠癌模型不同组织中的分布情况；
图18为AS1411-TP、TP、AS1411在荷瘤小鼠乳腺癌模型不同组织中的分布情况；
图19为AS1411-TP、TP、AS1411在荷瘤小鼠肺癌模型不同组织中的分布情况；
图20为AS1411-TP、TP、AS1411抗小鼠结肠癌异种植肿瘤图；
图21为AS1411-TP、TP、AS1411抗小鼠乳腺癌异种植肿瘤图；
图22为AS1411-TP、TP、AS1411抗小鼠肺癌异种植肿瘤图；
图23为AS1411-TP、TP、AS1411体内稳定性试验数据分析图；
具体实施方式
下面结合试验例及具体实施方式对本发明作进一步的详细描述。但不应将此理解为本发明上述主题的范围仅限于以下的实施例，凡基于本发明内容所实现的技术均属于本发明的范围。
实施例1
本实施例提供一种酸敏感性适配体雷公藤甲素偶联物，标记为Apt-TP，所述偶联物的结构式及反应路线如下所示：
适配子1是通过核酸合成仪AKTAOligopilot100system合成的氨基修饰的Apt；具体的，适配体1：NH2-(CH2)6-5’GCAGTTGATCCTTTGGATACCCTGG3’MS:7843；
具体的，提供上述实施例1提供的化合物5的制备过程：
化合物1：
在冰浴下，取1eq雷公藤甲素和8eq二甲基硫醚溶于乙腈溶液中，在40分钟内分四次加入4eq过氧化苯甲酰，氮气保护下将混合物在冰浴下搅拌1h，然后在室温下搅拌2小时。点板和LC-MS检测反应不在进行，混合物用乙酸乙酯稀释并用10％Na2CO3和盐水洗涤。有机相用无水Na2SO4干燥、过滤、浓缩得到粗品，将粗品过硅胶柱纯化得到纯品白色粉末化合物1(MS:420.2)；
化合物2：
将1eq化合物1溶于DCM溶液后，加入1.5eqSO2Cl2室温搅拌2h后，直接将溶剂旋干，得到粗品化合物2(MS:408.3)直接用于下一步；
化合物3：
将1.0eq的化合物2溶于20ml的DCM溶液中，加入0.7eq的18-冠醚-6和1.7eq的4-(烯丙氧基)-4-氧代丁酸钠，氮气保护下，常温搅拌2h，点板和LC-MS检测反应完全。再加入DCM稀释后，加入饱和碳酸氢钠，收集有机相后，用饱和食盐水洗两次，有机相用无水Na2SO4干燥、过滤、浓缩得到粗品，过硅胶柱纯化后得到化合物3(MS:530.3)；
化合物4：
将1.0eq的化合物3和2.5eq的1,3-二甲基巴比妥酸溶于体积比1:1的二氯甲烷和 乙酸乙酯溶液中，再加入0.1eq的四三苯基膦钯。整个反应体系保持在常温条件反应2h后，在40℃下减压除去有机溶剂，将所得到的粗品过柱纯化得到化合物4(MS：490.4)；
化合物5：
将合成好氨基修饰的Apt(适配体1)取1eq溶于去离子水中，加入100eq、PH为9的NaHCO3缓冲液，再加入100eq的DMT-MM水溶液，最后加入70eq溶于DMSO的化合物4。反应在摇床上37℃振荡24h后，HPLC监测反应完全后，加入2.5体积量的无水乙醇，将其搅匀放置干冰1h后取出低温下高速离心，收集沉淀用dd-H2O溶解成溶液，最后用RP-HPLC纯化收集产品再用冻干机冻干得到白色粉末纯品化合物5(MS:8527.3)。
实施例2
本实施例提供一种酸敏感性适配体雷公藤甲素偶联物，标记为E07-TP，所述偶联物的结构式及反应路线如下所示：
适配子2是通过核酸合成仪AKTAOligopilot100system合成的巯基PEG修饰的E07；
具体的，适配体2：
SH-CH2CH2-(OCH2CH2)10-O-5’GGACGGAUUUAAUCGCCGUAGAAAAGCAUGUCAAAGCCGGAACCGUCC3’MS:15990.5；
其中，偶联物7的制备过程包括如下步骤：
化合物1和化合物2与实施例1中的制备方法相同，此处不再进行赘述；
化合物6：
将1.0eq的化合物2溶于DCM溶液中，加入0.7eq的18-冠醚-6和2eq的3-马来酰亚胺基丙酸钠，氮气保护下，常温搅拌2h，点板和LC-MS检测反应完全。再加入DCM稀释后，加入饱和碳酸氢钠，收集有机相后，用饱和食盐水洗两次，有机相用无水Na2SO4干燥、过滤、浓缩得到粗品，过硅胶柱纯化后得到化合物6(MS:541.2)。
化合物7：
将1eq的巯基修饰E07(适配体2)溶于pH＝7.4的碳酸钠和碳酸氢钠的缓冲液中，加 入50eq的TECP活化,在4℃条件下振荡反应2h后，加入到溶有100eq的化合物6的DMF溶液中，反应体系保持在室温条件下反应24小时，反应结束后，2.5倍量乙醇沉淀，用RP-HPLC纯化粗品后得到目标产物化合物7(MS:16462.4)。
实施例3
本实施例提供一种酸敏感性适配体雷公藤甲素偶联物，标记AS1411-TP，所述偶联物的结构式及反应路线如下所示：
适配子3是通过核酸合成仪AKTAOligopilot100system合成的叠氮PEG修饰的AS1411；适配子3为
N3-CH2CH2-(OCH2CH2)3-O-5GGTGGTGGTGGTTGTGGTGGTGGTGG3’MS:8553.4
其中，偶联物7的制备过程包括如下步骤：
化合物1和化合物2与实施例1中的制备方法相同，此处不再进行赘述；
化合物8：
将1.0eq的化合物2溶于DCM溶液中，加入0.7eq的18-冠醚-6和3eq的4-戊炔酸钠，氮气保护下，常温搅拌2h，点板和LC-MS检测反应完全。再加入DCM稀释后，加入饱和碳酸氢钠，收集有机相后，用饱和食盐水洗两次，有机相用无水Na2SO4干燥、过滤、浓缩得到粗品，过硅胶柱纯化后得到化合物8(MS:470.4)。
化合物9：
将1eq叠氮PEG修饰的适配体3溶于pH＝7.4的碳酸钠和碳酸氢钠的缓冲液中，加入3eq的三乙胺和等体积叔丁醇，加入到溶有100eq的化合物8的DMF溶液中，再加入50eq的CuSO4.5H2O和50eq的抗血酸钠，氮气保护下反应体系室温反应24小时，反应结束后，2.5倍量乙醇沉淀，用RP-HPLC纯化粗品后得到目标产物化合物9(MS:9023.4)。
实施例1-3中制备得到的偶联物的MS图谱如图19-21所示。
一、偶合物的血清稳定性和弱酸微环境敏感性研究
为了验证本发明的偶合物的血清稳定性和弱酸微环境敏感性，分别进行了HPLC测 定，具体过程如下所述：
材料：AS1411-TP偶联物(缩醛酯键连接)、E07-TP偶联物(缩醛酯键连接)，AS1411-TP-1偶联物(烯醚键连接)，小鼠血清取自BALB/c小鼠，♀，7-8周龄(北京斯贝福生物技术有限公司)
实验方法：取AS1411-TP偶联物(缩醛酯键连接)、E07-TP偶联物(缩醛酯键连接)2mg溶解于100μL小鼠血清中，用于模拟偶联物在血液循环系统中稳定情况，平行设置6组。将药物与血清置于37℃培养箱中孵育，分别于0h、1h、2h、4h、12h、24h后将药物过滤，采用HPLC测定溶解不同时间后偶联物含量。测试结果见图4和图5；从图4和图5中可以看出，在24h内，HPLC图中仅存在偶联物，偶联物无分解现象，这表明本发明中的适配体-雷公藤甲素偶联物(缩醛键连接)可以在血清中长时间保持稳定。
肿瘤微环境是一种弱酸性环境，偶联物对弱酸微环境的敏感性决定了药物的治疗效果。取AS1411-TP-1偶联物(烯醚键连接)、AS1411-TP偶联物(缩醛酯键连接)、E07-TP偶联物(缩醛酯键连接)2mg溶解于100μL不同pH的PBS缓冲液中，用于模拟肿瘤微环境，2h后采用HPLC分别测定不同pH条件下偶联物的含量变化。其中，AS1411-TP偶联物(缩醛酯键连接)作为对照组，命名为AS1411-TP-1，AS1411-TP-1的化学式见图9所示；
检测结果见图6、图7和图8，图6为AS1411-TP偶联物的HPLC的测定图，图7为E07-TP偶联物的HPLC的测定图，图8为AS1411-TP-1的HPLC的测定图，从上述三幅图中可以看出通过缩醛酯键连接的偶联物在pH为6.5时便开始断裂，pH＝3.5为3.5时偶联物完全裂解，而通过烯醚键连接的偶联物在pH为3.5时才开始断裂，这证明缩醛酯键与烯醚键相比，断裂所需pH值更敏感，在肿瘤微环境中更容易断裂释放出出雷公藤甲素，这证明缩醛键连接的偶联物具有更好的肿瘤弱酸微环境的响应性。
上述血清稳定性和弱酸微环境敏感性实验充分说明本发明中偶联物中的缩醛酯键不仅具备血清稳定性，同时，在酸性环境下断键要求较低，当pH值为6左右，就能够实现连接键的断裂，释放雷公藤甲素，发挥药效。极大的提高了雷甲素的释放率，使得雷甲素能得到更加高效的利用。
核仁素适配体AS1411是一种高水溶性的单链的寡核苷酸，能够特异性与肿瘤细胞膜表面高表达的核仁素蛋白结合，因此以实施例3中核仁素适配体-雷公藤甲素偶联物为例，进行体外活性、体外毒性研究、体内分布、体内活性、体内稳定性等内容的研究，来充分的表征、验证本发明的偶联体中连接键的性能以及在体内抑制癌细胞的可行性。
二、AS1411-TP的体外活性研究
材料：适配体-雷公藤甲素偶联物(AS1411-TP)。雷公藤甲素(TP)由上海雅吉生物科技有限公司提供；适配体(AS1411)由成都先导药物开发股份有限公司获得；胰蛋白酶、噻唑蓝(MTT)(Biofroxx公司，德国)；二甲基亚砜(DMSO)(MPBiomedicals公司，法国)；胎牛血清(FBS)(Newzerum公司，新西兰)；DMEM培养基、RPMI1640培养基、培养皿、96孔板(康宁，美国)；结肠癌细胞HCT116、乳腺癌细胞MDA-MB-231、非小细胞肺癌细胞A549、胰腺癌细胞PANC-1、肝癌细胞HepG2均购自中国科学院上海细胞库并由本实验室保存。
实验过程：
2.1、细胞复苏
从液氮罐中取出肿瘤细胞株HCT116、MDA-MB-231、A549、PANC-1、HepG2，放置于37 ℃水浴锅中，待冻存细胞融化为细胞悬液后，以1000r/min的转速离心3min，弃去上清液，加入1ml新鲜且含10％胎牛血清和1％双抗的完全培养基将细胞混匀，再加入5ml培养基，用移液枪轻轻反复吹打细胞，使之混匀，最后将细胞悬液转移至10mm培养皿中，并标记好细胞名称，细胞代数，复苏日期和操作人的名字，放置于细胞培养箱中培养。(HCT116、PANC-1、HepG2细胞株采用DMEM培养基培养，MDA-MB-231细胞株和A549细胞株用RPMI1640培养基培养)。
2.2细胞传代
取出生长状态良好的HCT116、MDA-MB-231、A549、PANC-1、HepG2细胞，除去培养基，加入1ml无菌PBS洗净，加入1.5ml胰酶消化细胞，约2min后细胞变圆脱落，加入1.5ml培养基终止消化，转移至离心管，以1000r/min离心3min，弃去上清液，加入1ml新鲜培养基重悬，再加入新鲜培养基进行1：3比例分至其他培养皿，并标记传代日期和代数，放置于细胞培养箱中培养。
2.3 MTT实验测定AS1411-TP活性
取生长状态良好的HCT116、MDA-MB-231、A549、PANC-1、HepG2细胞，同细胞传代实验操作，细胞重悬后，用血球计数板进行细胞计数，以2500个/孔，种植于96孔板中，待细胞培养24h后，除去原来培养基，加入含不同浓度药物的培养基继续培养48小时。药物作用结束后，除去原来培养基，加入新鲜培养基100μl，加入10μl MTT溶液，置于培养箱中孵育2h后，加入150ml DMSO，摇床孵育15min，于490nm处，用酶标仪测定OD值，并计算细胞增殖抑制率。重复上述步骤三次。AS1411-TP对上述5种癌细胞系的抑制作用曲线图见图10-图14所示，实验数据见表1-5。
表1为AS1411-TP、TP、AS1411对结肠癌细胞系HCT116作用48h之后的细胞增殖抑制率的数据汇总表
表2为AS1411-TP、TP、AS1411对肺腺癌细胞系A549作用48h之后的细胞增殖抑制率的数据汇总表
表3为AS1411-TP、TP、AS1411对乳腺癌细胞系MDA-MB-231作用48h之后的细胞增殖抑制率的数据汇总表
表4为AS1411-TP、TP、AS1411对胰腺癌细胞系PANC-1作用48h之后的细胞增殖抑制率的数据汇总表
表5为AS1411-TP、TP、AS1411对肝癌细胞系HepG2作用48h之后的细胞增殖抑制率 的数据汇总表
结合上述表1-5的数据以及图3-7所示的药物对不同癌细胞的抑制作用统计折线图可以看出：雷公藤甲素对结肠癌细胞HCT116、乳腺癌细胞MDA-MB-231、肺腺癌细胞A549、胰腺癌细胞PANC-1、肝癌细胞HepG2的IC50分别为18.88±0.13nM、22.38±0.24nM、30.23±0.17nM、14.4±0.09nM、10.46±0.33nM；AS1411-TP对对结肠癌细胞HCT116、乳腺癌细胞MDA-MB-231、肺腺癌细胞A549、胰腺癌细胞PANC-1、肝癌细胞HepG2的IC50分别为20.01±0.12nM、20.03±0.27nM、29.87±0.16nM、11.18±0.03nM、11.21±0.26nM。在该实验浓度下，AS1411对各肿瘤细胞均无明显抑制作用。以上数据证明AS1411-TP对各肿瘤细胞均有与雷公藤甲素相当的抗肿瘤效果且非AS1411的作用，说明适配体的连接并不影响雷公藤甲素的抗肿瘤作用，这同时证明肿瘤微环境响应性酸性敏感连接键可在肿瘤细胞内断裂，释放出雷公藤甲素。
三、AS1411-TP的体外毒性研究
与上述第二部分中相同的材料此处不再赘述，除此以外还包括：人正常肝细胞LO2、人胚胎肾细胞HEK293均购自中国科学院上海细胞库并由本实验室保存。
实验过程
3.1细胞复苏
从液氮罐中取出肿瘤细胞LO2、HEK293细胞株，放置于37℃水浴锅中，待冻存细胞融化为细胞悬液后，以1000r/min的转速离心3min，弃去上清液，加入1ml新鲜且含10％胎牛血清和1％双抗的完全培养基将细胞混匀，再加入5ml培养基，用移液枪轻轻反复吹打细胞，使之混匀，最后将细胞悬液转移至10mm培养皿中，并标记好细胞名称，细胞代数，复苏日期和操作人的名字，放置于细胞培养箱中培养。(HEK293细胞株采用DMEM培养基培养，LO2细胞株用RPMI1640培养基培养)
3.2细胞传代
取出生长状态良好的LO2、HEK293细胞，小心吸去培养基，加入1ml无菌PBS洗净，加入1.5ml胰酶消化细胞，约2min后细胞变圆脱落，加入1.5ml培养基终止消化，转移至离心管，以1000r/min离心3min，弃去上清液，加入1ml新鲜培养基重悬，再加入新鲜培养基进行1：3比例分至其他培养皿，并标记传代日期和代数，放置于细胞培养箱中培养。
3.3 MTT实验测定适配体-雷公藤甲素偶联物细胞毒性
取生长状态良好的LO2、HEK293细胞，同细胞传代实验操作，细胞重悬后用血球计数板进行细胞计数，以3000个/孔，种植于96孔板中，待细胞培养24h后，除去原来培养基，加入含不同浓度药物的培养基继续培养48小时。药物作用结束后，去除掉原来的培养基，加入新鲜培养基100μl，加入10μlMTT溶液，置于培养箱中孵育2h后，加入150mlDMSO，摇床孵育15min，于490nm处，用酶标仪测定OD值，并计算细胞增殖抑制率。重复上述步骤三次。AS1411-TP对正常肝细胞LO2和正常人胚胎肾细胞HEK293的毒性曲线图见图15-16，实验数据见表6-7。
表6为AS1411-TP、TP、AS1411对人胚胎肾细胞HEK293作用48h之后的细胞增殖抑制率数据
表7为AS1411-TP、TP、AS1411对人正常肝细胞LO2作用48h之后的细胞增殖抑制率数据
结合上表6-7的实验数据以及图8-9所示的药物对正常肝细胞LO2和正常人胚胎肾细胞HEK293的细胞增殖抑制作用统计折线图可以看出：
雷公藤甲素对正常肝细胞LO2和正常人胚胎肾细胞HEK293具有较大的细胞毒性，其IC50分别为9.01±0.25nM、5.98±0.21nM，当雷公藤甲素偶联适配体AS1411后，其细胞毒性降低，偶联物对LO2和HEK293的IC50分别为51.86±0.13nM、48.10±0.13nM，AS1411对两种 正常细胞均无明显抑制作用。这充分证明适配体的连接降低了雷公藤甲素对正常细胞的细胞毒性。
其原因可能存在以下两个方面：1.适配体封闭了雷公藤甲素的活性基团，正常细胞为非弱酸性环境，雷公藤甲素无法完整释放。2.正常肝细胞和肾细胞细胞膜表面核仁素表达量较少，AS1411适配体与正常细胞不具靶向性，细胞对偶联物的摄入量较少，毒性作用小。
四、AS1411-TP的体内分布研究
为了验证AS1411-TP偶联物在体内的分布情况，分别对结肠癌、乳腺癌及肺癌小鼠模型进行体内分布实验，采用HPLC技术对测定其各组织中偶联物的含量，研究其体内分布规律，以下为不同荷瘤小鼠分别进行的体内分布研究。
4.1、结肠癌细胞系HCT116
材料：AS1411-TP给药组剂量设为50μM，给药体积为100μL。雷公藤甲素(TP)给药组剂量设为50μM，给药体积为100μL。与上述第二部分中相同的材料此处不再赘述，除此以外还包括实验动物：BALB/cnu小鼠，♀，6-7周龄。斯贝福(北京)生物技术有限公司提供；饲养于成都中医药大学实验动物房。
实验过程：在标准环境下饲养6-7周雌性裸鼠，将结肠癌细胞HCT116悬浮于无血清培养基中，每只小鼠接种3*106个肿瘤细胞。当肿瘤体积达到50-100mm3时，小鼠随机分为两组，分别尾静脉注射AS1411-TP、TP，每组12只。给药8h后处死动物，取出心、肝、脾、肺、肾以及肿瘤组织，用生理盐水冲洗干净，称重，-80℃短时冻存备用。取出各脏器组织，加入组织2倍量的生理盐水，匀浆后离心过滤，取90μL滤液，滤液中加入标准品溶液10μL，过滤后采用HPLC测定给药不同时间后各组织中药物含量。上述药物在荷瘤小鼠结肠癌模型不同组织中的分布情况见图17；
从图17中的分布结果显示，AS1411-TP在结肠癌肿瘤组织中分布较多，正常组织中分布较少，TP在正常肝、肾组织组织中分布较多。AS1411-TP在小鼠正常组织中含量远低于TP，而在肿瘤组织中，AS1411-TP实验组相对于雷公藤甲素实验组在肿瘤组织中的量占总注射量的百分比高，说明AS1411-TP在体内有较好的结肠癌肿瘤组织靶向性。
4.2、乳腺癌细胞系MDA-MB-231
本过程中材料与4.1中的材料相同的此处不再赘述，仅对癌细胞系进行了替换；
实验过程：在标准环境下饲养6-7周雌性裸鼠，将乳腺癌细胞MDA-MB-231悬浮于无血清培养基中，每只小鼠接种7*106个肿瘤细胞。当肿瘤体积达到50-100mm3时，小鼠随机分为两组，分别尾静脉注射适配体-雷公藤甲素偶联物、雷公藤甲素，每组12只。给药4h后处死动物，取出心、肝、脾、肺、肾以及肿瘤组织，用生理盐水冲洗干净，称重，-80℃短时冻存备用。用HPLC检测组织中的适配体-雷公藤甲素以及雷公藤甲素的含量。取出各脏器组织，加入组织2倍量的生理盐水，匀浆后离心过滤，取90μL滤液，滤液中加入标准品溶液10μL，过滤后采用HPLC测定给药不同时间后各组织中药物含量。上述药物在荷瘤小鼠乳腺癌模型不同组织中的分布情况见图18；
从图18的分布情况可以看出：AS1411-TP在乳腺癌肿瘤组织中分布较多，正常组织中分布较少，雷公藤甲素在正常肝、肾组织组织中分布较多。AS1411-TP在小鼠正常组织中含量远低于雷公藤甲素；而在肿瘤组织中，AS1411-TP实验组在肿瘤组织中的量占总注射量 的百分比高于雷公藤甲素实验组，说明AS1411-TP在体内有较好的乳腺癌肿瘤组织靶向性。
4.3、肺癌细胞系A549
本过程中材料与4.2中的材料相同的此处不再赘述，仅对癌细胞系进行了替换；
实验方法：在标准环境下饲养6-7周雌性裸鼠，将肺癌细胞A549悬浮于无血清培养基中，每只小鼠接种6*106个肿瘤细胞。当肿瘤体积达到50-100mm3时，小鼠随机分为两组，分别尾静脉注射适配体-雷公藤甲素偶联物、雷公藤甲素，每组12只。给药4h后处死动物，取出心、肝、脾、肺、肾以及肿瘤组织，用生理盐水冲洗干净，称重，-20℃短时冻存备用。用HPLC检测组织中的AS1411-TP以及TP的含量。用HPLC检测组织中的适配体-雷公藤甲素偶联物以及雷公藤甲素的含量。取出各脏器组织，加入组织2倍量的生理盐水，匀浆后离心过滤，取90μL滤液，滤液中加入标准品溶液10μL，过滤后采用HPLC测定给药不同时间后各组织中药物含量。上述药物在荷瘤小鼠肺癌模型不同组织中的分布情况见图19；
从图19中可以看出AS1411-TP在肿瘤组织中分布较多，正常组织中分布较少，雷公藤甲素正常肝、肾组织组织中分布较多。AS1411-TP在小鼠正常组织中含量远低于雷公藤甲素，而在肿瘤组织中，AS1411-TP实验组相对于雷公藤甲素实验组在肿瘤组织中的量占总注射量的百分比高，说明AS1411-TP在体内有较好的肺癌细胞A549肿瘤靶向性，同时规避其他组织，达到肿瘤靶向的效果。
五、AS1411-TP的体内活性实验研究
为了验证AS1411-TP偶联物的体内抗肿瘤作用，分别对结肠癌、乳腺癌及肺癌小鼠模型进行体内抗肿瘤实验，对其肿瘤肿瘤、RTV值、肿瘤抑制率及T/C值进行测定，验证其抗肿瘤作用。以下即为不同荷瘤小鼠分别进行的体内活性研究：
5.1、结肠癌细胞系HCT116
在标准环境下饲养6-7周雌性裸鼠，将结肠癌细胞HCT116悬浮于无血清培养基中，每只小鼠接种5*106个肿瘤细胞。当肿瘤体积达到50-100mm3时，小鼠随机分为4组，分别尾静脉注射适配体-雷公藤甲素偶联物、雷公藤甲素、适配体，每组12只。设PBS阴性对照组。给药组：TP(10μM、30μM及50μM，给药体积为100μL)，AS1411(10μM、30μM及50μM，给药体积为100μL)以及AS1411-TP(10μM、30μM及50μM，给药体积为100μL)。每2天给药1次，连续给药8次，在末次给药后24小时结束实验。实验结束后将动物断颈椎处死，剥离肿瘤，称瘤重，计算药物对肿瘤生长抑制率。用t检验法比较各组动物肿瘤重量、肿瘤体积、RTV等指标。实验数据汇总于表8，图20展示出不同注射药物得到的抗小鼠结肠癌异种植肿瘤。
表8为AS1411-TP、TP、AS1411对荷瘤小鼠结肠癌肿瘤模型生长抑制作用实验数据汇总表
在实验观察过程中，TP、AS1411-TP、AS1411各组在给药8次期间小鼠体重基本维持在动物毒副反应可以耐受的范围。PBS阴性对照组小鼠体重呈缓慢逐渐递减势，与分组开始时相比，平均体重减少1.7g。由动物肿瘤重量、肿瘤抑制率及RTV值可知，TP组、AS1411-TP组、AS1411组各给药组小鼠肿瘤生长速度和肿瘤抑制率与PBS组相比有所减缓且呈剂量依赖关系。其中，AS1411-TP组和TP组给药对动物肿瘤增长速度、肿瘤抑制率呈明显剂量反应关系。但所有指标均显示出，TP组肿瘤抑制作用均低于相应浓度的AS1411-TP组。
实验结论：AS1411-TP 10μM、30μM及50μM对结肠癌模型裸鼠尾连续8次静脉注射给药，肿瘤生长受到明显抑制，且抑制作用优于TP同浓度下给药，说明AS1411-TP在体内具有较好的抗肿瘤作用。
5.2、乳腺癌细胞系MDA-MB-231
实验所用的材料与5.1结肠癌细胞系测试的材料一致，其区别仅在于瘤株不同；
具体实验方法：在标准环境下饲养6-7周雌性裸鼠，将乳腺癌细胞系MDA-MB-231悬浮于无血清培养基中，每只小鼠接种6*106个肿瘤细胞。当肿瘤体积达到50-100mm3时，小鼠随机分为4组，分别尾静脉注射适配体-雷公藤甲素偶联物、雷公藤甲素、适配体，每组12只。设PBS阴性对照组。给药组：雷公藤甲素(10μM、30μM及50μM)，AS1411(10μM、30μM及50μM)以及AS1411-TP(10μM、30μM及50μM)。每2天给药1次，连续给药8次，在末次给药后24小时结束实验。实验结束后将动物断颈椎处死，剥离肿瘤，称瘤重，计算药物对肿瘤生长抑制率。用t检验法比较各组动物肿瘤重量、肿瘤体积、RTV等指标。实验数据汇总于表9，图21展示出不同注射药物得到的抗小鼠乳腺癌异种植肿瘤图。
表9：AS1411-TP、TP、AS1411对荷瘤小鼠乳腺癌模型生长抑制作用实验数据汇总表
在实验观察过程中，TP、AS1411-TP、AS1411各组在给药8次期间小鼠体重基本维持在动物毒副反应可以耐受的范围。PBS阴性对照组小鼠体重呈缓慢逐渐递减势，与分组开始时相比，平均体重减少2.1g。由动物肿瘤重量、肿瘤抑制率及RTV值可知，TP组、AS1411-TP、AS1411各给药组小鼠肿瘤生长速度和肿瘤抑制率与PBS组相比有所减缓且呈剂量依赖关系。其中，AS1411-TP和TP组给药对动物肿瘤增长速度、肿瘤抑制率呈明显剂量反应关系。但所有指标均显示出，TP组肿瘤抑制作用均低于相应浓度的AS1411-TP组。
通过表9和图14对应的显示结果，可以得出：AS1411-TP 10μM、30μM及50μM连续8次对乳腺癌裸鼠模型尾静脉注射给药，乳腺癌肿瘤细胞生长受到明显抑制，抑制效率与给药剂量明显相关，且抑制作用优于TP同浓度下给药，说明AS1411-TP对乳腺癌具有较好体内效果。
5.3、肺癌细胞系A549
实验方法：在标准环境下饲养6-7周雌性裸鼠，将肺癌细胞系A549悬浮于无血清培养基中，每只小鼠接种6*106个肿瘤细胞。当肿瘤体积达到50-100mm3时，小鼠随机分为4组，分别尾静脉注射AS1411-TP、雷公藤甲素、适配体，每组12只。设PBS阴性对照组。给药组TP(10μM、30μM及50μM)，AS1411(10μM、30μM及50μM)以及AS1411-TP(10μM、30μM及50μM)。每2天给药1次，连续给药8次，在末次给药后24小时结束实验。实验结束后将动物断颈椎处死，剥离肿瘤，称瘤重，计算药物对肿瘤生长抑制率。用t检验法比较各组动物肿瘤重量、肿瘤体积、RTV等指标。实验数据汇总于表10，图22展示出不同注射药物得到的抗小鼠肺癌异种植肿瘤。
表10为AS1411-TP、TP、AS1411对荷瘤小鼠肺腺癌肿瘤模型生长抑制作用实验数据汇总表
在实验观察过程中，TP、AS1411-TP、AS1411各组在给药8次期间小鼠体重基本维持在动物毒副反应可以耐受的范围。PBS阴性对照组小鼠体重呈缓慢逐渐递减势，与分组开始时相比，平均体重减少2.0g。由动物肿瘤重量、肿瘤抑制率、RTV值等可知，TP组、AS1411-TP、AS1411各给药组小鼠肿瘤生长速度和肿瘤抑制率与PBS组相比有所减缓且呈剂量依赖关系。其中，AS1411-TP和TP组给药对动物肿瘤增长速度、肿瘤抑制率呈明显剂量反应关系。但所有指标均显示出，TP组肿瘤抑制作用均低于相应浓度的AS1411-TP组。
通过表10和图22对应的显示结果，可以得出：AS1411-TP 10μM、30μM及50μM连续8次对肺腺癌细胞系A549裸鼠尾静脉注射给药，肺癌细胞系A549肿瘤生长受到明显抑制，抑制效率与给药剂量明显相关，且优于TP同浓度下给药，说明AS1411-TP对肺腺癌具有较好的靶向抑制效果。
六、AS1411-TP体内稳定性研究
通过对适配体雷公藤甲素偶联物进行小鼠体内的稳定性实验，来模拟药物在人体内的稳定性，以验证该偶联物在不同组织中的解离度和稳定性。
实验方法：随机挑选27只荷瘤小鼠，于尾椎静脉注射浓度为50μMAS1411-TP的生理盐水溶液100μL。分别在0.5、1、2、4、8、12、16、24、32h时，各处死3只老鼠，腹腔注射戊巴比妥钠麻醉，采用心脏穿刺取血，取心、肝、脾、肺、肾、实体瘤等组织，进行组织匀浆、提取，样品处理及预实验分别确定TP、AS1411及AS1411-TP偶联物的保留时间后，HPLC分别检测每个时间点各组织中游离的TP和AS1411-TP的量，并计算出释放效率，监测并定量考察药物在组织内的稳定性。实验数据见表11，体内稳定性试验数据分析结果见图23。
表11为适配体雷公藤甲素偶联物(AS1411-TP)物体内稳定性试验数据
如图23为适配体雷公藤甲素偶联物(AS1411-TP)体内稳定性试验数据分析图，通过表11与图16的数据，可以显示出：AS1411-TP偶联物经过36小时后在血浆及心、脾，肺等正常组织中几乎不解离或仅少量解离，在肾和肝组织中解离稍多；而在肿瘤组织中，AS1411-TP偶联物大量解离，并且随着时间的累积，解离度不断增加。以上结果证明了用于连接雷公藤甲素和适配体的连接键在体内起到了明显的作用。使得偶联物在正常组织中不发生断裂，毒性较小；而在肿瘤组织中断裂，释放出雷公藤甲素，发挥治疗作用。
总结：综合上述对药物的各方面实验研究后，本发明提供的适配体雷公藤甲素偶联物是以缩醛酯官能团为连接键。该连接键与已有的烯醚键相比，其具有更好的弱酸响应性，断链pH为3.5-6.5，而烯醚键的断裂条件为pH＝3.5以下，因此针对肿瘤微环境，本发明提供的适配体雷公藤甲素偶联物更具有肿瘤微环境响应性。同时，体外实验证明，本发明提供的适配体雷公藤甲素偶联物具有较好的血清稳定性。
本发明提供的适配体雷公藤甲素偶联物在体内外均具有与原药相当的抗肿瘤效果，这证明适配体的连接并不影响偶联物的抗肿瘤作用。同时，体内外实验均表明与原药相比，偶联物毒性降低，这证明偶联物在具有较好的靶向抗肿瘤作用，同时可规避对正常组织的毒性。
本发明提供的适配体雷公藤甲素偶联物在肿瘤组织中的分布远高于其它组织，同时在肿瘤组织中的解离效率远高于正常组织，这证明本发明提供的偶联物在体内具有较好的肿瘤靶向性，同时，肿瘤微环境可满足偶联物的释放条件。
以上所述仅是本发明的优选实施方式，应当理解本发明并非局限于本文所披露的形式，不应看作是对其他实施例的排除，而可用于各种其他组合、修改和环境，并能够在本文所述构想范围内，通过上述教导或相关领域的技术或知识进行改动。而本领域人员所进行的改动和变化不脱离本发明的精神和范围，则都应在本发明所附权利要求的保护范围内。

Claims (10)

  1. 一种酸敏感性适配体雷公藤甲素偶联物，其特征在于，所述偶联物通式如下：
    其中，A为核酸适配体；
    B为连接雷公藤甲素与核酸适配体的连接键，所述连接键在雷公藤甲素的第14位羟基位置形成有缩醛酯官能团。
  2. 一种酸敏感性适配体雷公藤甲素偶联物，其特征在于，所述偶联物通式如下式所示：
    其中，A为核酸适配体；
    B为连接雷公藤甲素与核酸适配体的连接键，所述连接键在雷公藤甲素的第14位羟基位置形成有缩醛酯官能团。
  3. 根据权利要求1或2所述的酸敏感性适配体雷公藤甲素偶联物，其特征在于，所述连接键，即B的通式结构如下：
  4. 根据权利要求3所述的酸敏感性适配体雷公藤甲素偶联物，其特征在于，B1的结构通式为
    其中，0≤n1≤100，优选的，0≤n1≤50，更优选的，0≤n1≤20；
    其中，0≤n2≤100，优选的，0≤n2≤50，更优选的，0≤n2≤20；
    n1和n2的取值范围包括0，但不同时为0；
    具体的，n1、n2取值范围优选为2、3、5、10。
  5. 根据权利要求3所述的酸敏感性适配体雷公藤甲素偶联物，其特征在于，
    B2的通式如下：
  6. 根据权利要求1所述的酸敏感性适配体雷公藤甲素偶联物，其特征在于，所述核酸适配体是经过修饰的核酸适配体，所述经过修饰的核酸适配体结构通式为A1-C1；其中，C1的结构为：
    其中，0≤n3≤100，优选的，0≤n3≤50，更优选的，0≤n3≤20；
    其中，0≤n4≤100，优选的，0≤n4≤50，更优选的，0≤n4≤20；
    n3和n4的取值范围包括0，但不同时为0；
    具体的，n3、n4取值范围优选为2、3、5、10。
  7. 根据权利要求6所述的酸敏感性适配体雷公藤甲素偶联物，其特征在于，A1是AS1411、Pegaptanib、Sgc8c、A10、DNAaptamer、RNAaptamer、CL4、Apt和E07中的任意一种。
  8. 根据权利要求7所述的酸敏感性适配体雷公藤甲素偶联物，其特征在于，
    A1的序列如下：
    AS1411：GGTGGTGGTGGTTGTGGTGGTGGTGG；
    Pegaptanib：GCGAACCGAUGGAAUUUUUGGACGCUCGC；
    Sgc8c：ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA；
    A10：GGGAGGACGAUGCGGAUCAGCCAUGUUUACGUCACUCCUUGUCAAUCCUCAUCGGCAGACGACUCGCC
    CGA；
    DNA aptamer：GGGAGACAAGAATAAACGCTCAA-(25N)-TTCGACAGGAGGCTCACAACAGGC；
    RNA aptamer：GGGGGCAUACUUGUGAGACUUUUAUGUCACCCCC；
    CL4：GCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAGGC；
    Apt：GCAGTTGATCCTTTGGATACCCTGG；
    E07：GGACGGAUUUAAUCGCCGUAGAAAAGCAUGUCAAAGCCGGAACCGUCC。
  9. 一种根据权利要求8所述的酸敏感性适配体雷公藤甲素偶联物的制备方法，其特征在于，包括以下步骤制备得到：
    步骤1：制备雷公藤甲素衍生物，所述雷公藤甲素衍生物中，雷公藤甲素的第14位羟基位置连接有缩醛酯官能团；
    步骤2：对核酸适配体进行修饰得到经过修饰的核酸适配体修饰体A1-C1；
    步骤3：将雷公藤甲素衍生物与步骤2中的核酸适配体修饰物通过B2连接键连接即得酸敏感性适配体雷公藤甲素偶联物。
  10. 如权利要求1或2中所述的酸敏感性适配体雷公藤甲素偶联物在制备抗肿瘤药物中的应用。
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