Tetrodoxin conjugate and its preparation method and application

Abstract

Tetrodoxine conjugate and its preparation method and application. A synthetic carboxyl-containing polymer compound and tetrodoxine are used as raw materials, a catalyst and a solvent are added, and a tetrodoxine conjugate is obtained through a coupling reaction. The carboxyl-containing polymer compound is selected from carboxylated dextran or carboxylated β-cyclodextrin. The tetrodoxine conjugate can be used to prepare sodium channel blockers and drugs for treating neuropathic pain caused by chemotherapy. The tetrodoxine conjugate of the present invention has analgesic efficacy associated with TTX, and the drug window is enlarged, and the safety of medication is greatly improved.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a tetrodoxine conjugate, a preparation method of the tetrodoxine conjugate, and an application of the tetrodoxine conjugate. Background Art

[0002] Tetrodotoxin (TTX, CAS: 4368-28-9), also known as Halneuron, Tectin or Tetrodin, is an aminoperhydroquinazoline compound with a molecular formula of C 11 H 17 O8N3, molecular weight is 319.27, its molecular structure is as follows:

[0003]

[0004] Tetrodoxin is an alkaloid and one of the most toxic neurotoxins found in nature. It was once considered the most toxic non-protein toxin in nature and is commonly found in pufferfish and other organisms. Japanese scientist Yoshizumi Tahara first isolated Tetrodoxin in 1909; in 1955, Hirata Yoshimasa successfully isolated Tetrodoxin from pufferfish, and then realized the artificial synthesis of Tetrodoxin in 1972.

[0005] As a typical sodium channel blocker, the mechanism of action of tetrodoxin can be simply described as: when tetrodoxin passes through the voltage-gated sodium channel, the hydroxyl group and guanidine group on tetrodoxin form hydrogen bonds with the amino acid residues on the channel, so that the voltage-dependent sodium ion channel is blocked, thereby affecting a series of physiological functions such as nerve excitation and conduction, the regulatory function of the central nervous system, and heart beat, smooth muscle peristalsis, skeletal muscle contraction, and hormone secretion of the animal, leading to muscle and nerve paralysis. Its mechanism of action includes Nav1.7 blockers, Nav1.8 blockers, and Nav1.5 blockers. The industry believes that tetrodoxin is widely used in the fields of nervous system diseases, cancer pain, neuralgia, anesthesia, heroin dependence, etc. However, the problems existing in the use of tetrodoxin in the prior art have not been properly solved, which limits the application of tetrodoxin. The application direction currently being carried out is mainly to relieve the pain of cancer patients.

[0006] The anesthetic effect of tetrodoxin is stronger than that of commonly used anesthetics. It can exert a strong analgesic and anesthetic effect in a trace amount in the body. Not only does it have a long-lasting anesthetic effect, but it can also block the withdrawal symptoms induced by the opioid naloxone after morphine withdrawal. The main disadvantage of tetrodoxin as an anesthetic is its poor solubility. It is only slightly soluble in weak acids, anhydrous ethanol, and ether. In addition, its high activity, high toxicity, and low therapeutic index seriously affect its clinical research and application. In particular, the safety and stability of tetrodoxin have always been the main obstacles that are difficult to solve. Due to the inability to determine the safe dose and the effective dose that passes the blood-brain barrier, the application of monomer tetrodoxin in the field of analgesia has not been reported. When the dose is too large, it can cause severe hypotension and respiratory depression; when the dose is too small, it cannot produce analgesic effects (as described in reference 1).

[0007] In order to overcome the above-mentioned shortcomings of tetrodoxin, technicians in this field continue to develop tetrodoxin derivatives. Unfortunately, various technical routes have their own shortcomings, including high burst drug release, low mechanical strength of gel leading to potential dose dumping, poor biocompatibility of polymer micelle system and gradual reduction of system pH value due to acidic degradation, and potential safety hazards of human body, environment and organisms caused by irritating small molecule acyl chloride raw materials (such as literature 2, literature 3, literature 4 and literature 5). Although the development of tetrodoxin conjugates with natural compound molecules has the advantage of higher safety after hydrolysis, the number of natural compound molecules available is small. The development of tetrodoxin conjugates with artificial synthetic molecules has the advantage of a wider range of selectable molecules. However, there are no relevant research results on the application of tetrodoxin conjugates in tetrodoxin pharmaceutical technology.

[0008] Compared with synthetic polymer groups, natural polymer groups interact better with functional groups on the cell surface, have higher molecular structure stability, are renewable and biodegradable, and natural polymer materials also have good biocompatibility, that is, they can interact with organisms without obvious toxicity. However, natural polymer materials also have some limitations. First, their performance is relatively poor. Compared with synthetic polymer materials, the mechanical properties, thermal stability and chemical resistance of natural polymer materials need to be improved. Secondly, the preparation process of natural polymer materials is complicated and costly. The process of extracting and modifying natural organic matter requires a lot of energy and chemical reagents, and has high technical requirements. In short, the performance and preparation process of natural polymer materials still face some challenges, which need to be solved through in-depth research and technological improvements. In contrast, although synthetic polymer groups may carry a certain proportion of isomers, and chemical photoinitiators, crosslinkers, surfactants and other substances that are toxic to organisms may be used in the synthesis process, these potential risks can be reduced through reasonable design and optimization. In addition, synthetic polymer groups have a wider range of molecular selectivity and controllability, and their structure and properties can be adjusted as needed to meet specific drug delivery and release requirements. Therefore, the application of synthetic polymer groups in the pharmaceutical field still has its significance and value.

[0009] Reference 1: Liu Min, Zhao Chuangxin, Qi Xiuli, et al. Distribution and metabolic dynamics of tetrodotoxin in mice [J]. Chinese Pharmacological Bulletin, 2006, 22 (4): 507-508.

[0010] Document 2: Ruel-Gariepy E, Leroux J C. In situ-forming hydrogels—review of temperature-sensitive systems[J]. European Journal of Pharmaceutics andBiopharmaceutics, 2004, 58(2): 409-426.

[0011] Document 3: Schmaljohann D. Thermo-and pH-responsive polymers in drug delivery[J]. Advanced drug delivery reviews, 2006, 58(15): 1655-1670.

[0012] Document 4: Wex Pharmaceuticals Co., Ltd. Use of sodium channel blockers in the preparation of drugs for treating neuropathic pain caused by chemotherapy: CN200780011388.3[P], 2012-12-05.

[0013] Reference 5: Fujian Zhangping Daisy Biopharmaceutical Co., Ltd. A low-toxic tetrodotoxin ester derivative with analgesic effect and its preparation method and application: CN202311187063.8[P], 2024-09-10. Summary of the invention

[0014] In order to solve the shortcomings of the current pharmaceutical technology of tetrodoxin, one of the purposes of the present invention is to provide a tetrodoxin conjugate (PTTX for short), the general formula of which is:

[0015] ; wherein R' is the skeleton of an artificially synthesized carboxyl-containing polymer compound; the carboxyl-containing polymer compound is selected from carboxylated dextran or carboxylated β-cyclodextrin.

[0016] Preferably, the structural formula of the tetrodoxin conjugate is selected from formula (I) or (II):

[0017] (I), n=1-20000 in formula (I);

[0018] (II), wherein R=COCH2CH2COOH or H.

[0019] The tetrodoxin conjugate of formula (I) is hereinafter referred to as PTTX1; the tetrodoxin conjugate of formula (II) is hereinafter referred to as PTTX2.

[0020] The second object of the present invention is to provide a method for preparing the tetrodoxin conjugate, which uses an artificially synthesized carboxyl-containing polymer compound and tetrodoxin as raw materials, adds a catalyst and a solvent, and then obtains the tetrodoxin conjugate through a coupling reaction. The carboxyl-containing polymer compound is selected from carboxylated dextran or carboxylated β-cyclodextrin.

[0021] Preferably, the catalyst for the coupling reaction is selected from any one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N,N'-dicyclohexylcarbodiimide, and a combination of N,N-dimethylaminopyridine or other organic bases.

[0022] Preferably, the solvent for the coupling reaction is selected from non-hydroxyl solvents, including but not limited to any one of dimethyl sulfoxide or N,N-dimethylformamide.

[0023] The third object of the present invention is to provide the use of the tetrodotoxin conjugate in the preparation of sodium channel blockers and drugs for treating neuropathic pain caused by chemotherapy.

[0024] The fourth object of the present invention is to provide a tetrodoxin conjugate preparation, which is prepared by adding a pharmaceutically acceptable excipient to the tetrodoxin conjugate. The tetrodoxin conjugate preparation can be hydrolyzed and release tetrodoxin after entering the body. Different tetrodoxin conjugate preparations have different drug windows.

[0025] Preferably, the pharmaceutically acceptable excipients include inert organic carriers and / or inorganic carriers. For example, the excipients are specifically selected from at least one of water, saline, acetic acid, gelatin, gum arabic, lactose, starch, cellulose, magnesium stearate, talc, vegetable oil, cyclodextrin, polyalkylene glycol, etc.

[0026] Preferably, the dosage form of the tetrodotoxin conjugate preparation is an injection.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] The tetrodotoxin conjugate of the present invention has analgesic efficacy associated with TTX, and the selection of polymer conjugation increases the effective amount of the drug, increases the drug window, and greatly improves the safety of medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the general formula of the tetrodotoxin conjugate of the present invention.

[0030] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of dextran raw material in D2O.

[0031] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of carboxylated dextran raw material in D2O.

[0032] Figure 4 This is the H NMR spectrum of TTX in 4%CD3COOD / 96%D2O. 1 H NMR (400 MHZ, 4%CD3COOD / 96%D2O) δ: 5.45 (d, J = 9.4 HZ, 1H), 4.26 (d, J = 1.8 HZ, 1H), 4.22 (br, 1H), 4.05 (t, J = 2.1 HZ, 1H), 3.99~4.01 (m, 2H), 3.93 (s, 1H), 2.33 (d, J = 9.5 HZ, 1H).

[0033] Figure 5 This is the H NMR spectrum of PTTX1 in D2O, and the characteristic peaks of TTX can be observed.

[0034] Figure 6 Particle size distribution of PTTX1 in water (0.16 g / L).

[0035] Figure 7 In vitro release curves of different TTX conjugates, where: Figure 7 A is the in vitro release curve of PTTX1 in PBS (pH = 7.4); Figure 7 B is the in vitro release curve of PTTX2 in PBS (pH = 7.4); Figure 7 C is the in vitro release curve of TDP-TTX in PBS (pH=7.4) (derived from reference Zhao C, Liu A, Santamaria CM, et al. Polymer-tetrodotoxin conjugates to induce prolonged duration local anesthesia withminimal toxicity[J]. Nature communications, 2019, 10(1): 2566.).

[0036] Figure 8 Cell viability of PC12 cells incubated in different concentrations of carboxylated dextran for 24 hours. The ordinate represents the cell viability (the ratio of cell viability at different polymer concentrations to that of blank control cells) (100%), and the abscissa represents the concentration of carboxylated dextran (w / w).

[0037] Fig. 9 Cell viability of C2C12 cells incubated in different concentrations of carboxylated dextran for 24 hours, where the ordinate represents cell viability (100%) and the abscissa represents the concentration of carboxylated dextran (w / w).

[0038] Fig.10 It is a living color chromosome-carboxylated glucan map.

[0039] Fig.11 Cell viability of PC12 cells incubated in different concentrations of carboxylated β-cyclodextrin for 24 hours, where the ordinate represents cell viability (100%) and the abscissa represents the concentration of carboxylated β-cyclodextrin (w / w).

[0040] Fig.12 Cell viability of C2C12 cells incubated in different concentrations of carboxylated β-cyclodextrin for 24 hours, where the ordinate represents cell viability (100%) and the abscissa represents the concentration of carboxylated β-cyclodextrin (w / w).

[0041] Fig.13This is a live color chromosome-carboxylated β-cyclodextrin map.

[0042] Fig.14 The gross morphological changes of MADB106 cells inoculated into the rat plantar surface. DETAILED DESCRIPTION

[0043] The following is a further detailed description of the above contents of the present invention through specific implementation methods in the form of embodiments. However, it should not be understood that the scope of the above subject matter of the present invention is 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.

[0044] The tetrodoxin conjugate used in the present invention was prepared by the applicant. The remaining experimental materials were all from commercial sources unless otherwise specified. Polyvinyl alcohol was purchased from Aladdin, dextran, β-cyclodextrin, dimethyl sulfoxide, N,N-dimethylaminopyridine, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride were all purchased from Anergy, tetrodoxin was from Huamu, and TTX Elisa kit was from Reagen. The abbreviations used in the description of this application are as follows,

[0045] Dimethyl sulfoxide: DMSO,

[0046] N,N-dimethylaminopyridine: DMAP,

[0047] 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride: EDC . HCl,

[0048] Tetrodotoxin: TTX,

[0049] Tetrodoxin conjugate: PTTX (its general structural formula is as follows Figure 1 as shown).

[0050] In this application, 0.6% acetic acid aqueous solution is prepared as follows: accurately measure 0.6 mL of acetic acid, add normal saline for injection, make up to 100 mL, and shake well to obtain 0.6% acetic acid aqueous solution (prepare and use immediately).

[0051] Example 1

[0052] Step 1, preparing PTTX-1 (PTTX1), the synthetic route is as follows:

[0053]

[0054] Step 1.1, preparation of carboxylated dextran.

[0055] The reaction materials and dosages include: 555.5 mg succinic anhydride, 205.6 mg dextran (molar mass: 20000 Da, its nuclear magnetic resonance hydrogen spectrum is as follows Figure 2As shown), 91.7 mg N,N-dimethylaminopyridine.

[0056] In a nitrogen atmosphere, the above raw materials were dissolved in anhydrous dimethyl sulfoxide, stirred at room temperature overnight, dialyzed and freeze-dried (-40°C, 72h), and a white solid product was obtained, which was carboxylated dextran (its H NMR spectrum was as follows Figure 3 as shown).

[0057] Step 1.2, preparation of PTTX1.

[0058] The reaction materials and amounts include: 77.2 mg (prepared in step 1.1) carboxylated dextran, 12.0 mg EDC . HCl, 0.64 mg DMAP, 8.0 mg TTX.

[0059] In a nitrogen atmosphere, TTX was dissolved and dispersed in anhydrous DMSO in a reaction bottle, and then other raw materials were added and reacted at room temperature for one week. After the reaction, the reaction solution was dialyzed and freeze-dried (-40°C, 72h) to obtain the product as a white solid PTTX1, which weighed 71.54 mg after washing and drying, and the reaction yield was greater than 81.5%.

[0060] The H NMR spectrum of PTTX1 is shown in Figure 5 As shown, it can be observed that TTX (its H NMR spectrum is shown in Figure 4 Weigh an appropriate amount of PTTX1 and disperse it evenly in water to prepare a 0.16 g / L solution. The particle size distribution is shown in Figure 6 shown.

[0061] Step 2: Prepare the preparation stock solution of PTTX1.

[0062] 40 mg of PTTX1 prepared in step 1 of this example was accurately weighed and then dissolved in physiological saline to a fixed concentration of 20 g / L to obtain a PTTX1 preparation mother solution, which was stored at -20°C for future use.

[0063] Example 2

[0064] Step 1, prepare PTTX2 conjugate-2 (PTTX2), the synthetic route is as follows:

[0065]

[0066] Step 1.1 Preparation of carboxylated β-cyclodextrin.

[0067] The reaction raw materials and dosages include: 1.0 g of β-cyclodextrin, 2.8 g of succinic anhydride, and 0.45 g of N,N-dimethylaminopyridine.

[0068] In a nitrogen atmosphere, add the above raw materials to the reaction bottle, dissolve with 10 ml of anhydrous DMSO, and stir overnight at room temperature. After the reaction is completed, dialyze the reaction solution for 24 hours using a dialysis bag (molar mass: 500Da). The dialysis fluid is deionized water with a volume of 3L, and the water is changed 4 times. After the dialysis is completed, freeze-dry (-40°C, 72h) to obtain a white solid product, which is carboxylated β-cyclodextrin.

[0069] Step 1.2 Preparation of PTTX2.

[0070] The reaction materials and dosages include: 53 mg (prepared in step 1.1) carboxylated β-cyclodextrin, 1.0 mg TTX, 6.9 mg EDC . HCl, 2.6 mg DMAP.

[0071] In a nitrogen atmosphere, the above raw materials were added to the reaction bottle, dissolved with 5 ml of anhydrous DMSO, and reacted at room temperature for 7 days. After the reaction, the reaction solution was dialyzed with a dialysis bag (Mw: 1000Da) for 24 hours against water. The dialysis fluid was deionized water, the dialysis fluid volume was 3L, and the water was changed 4 times. After the dialysis, freeze-dried (-40°C, 72h) was obtained to obtain a white solid PTTX2, which weighed 45.00 mg after rinsing and drying, and the reaction yield was greater than 83%.

[0072] Step 2: Preparation of Tetradoxine Conjugate-2 (PTTX2) Preparation Stock Solution

[0073] 5.0 mg of PTTX2 prepared in step 1 of this example was accurately weighed and then dissolved in physiological saline to a fixed concentration of 5 g / L to obtain a PTTX2 preparation mother solution, which was stored at -20°C for future use.

[0074] Comparative Example 1

[0075] Synthesis of TDP-TXX drug conjugates:

[0076] Dry polyethylene glycol (0.005 mol) and sebacic acid (2.02 g, 0.01 mol) were added to a round-bottom flask. 8 mL of anhydrous DMF, 8 mL of anhydrous DMSO, and 4 mL of anhydrous DCM were added. After sonication for 5-10 minutes, DIC (4.336 mL, 28 mmol) and DMAP (0.489 g, 4 mmol) were added. Glycerol (184 μL, 0.0025 mol) was added and allowed to stand at room temperature for 24 h. Anhydrous DMSO (10 mL) solution with TTX (1 mg, 0.003 mmol) was added, and the mixture was allowed to stand at room temperature for 7 days. After the reaction was completed, DCM was removed from the reaction mixture using a rotary evaporator. The residue was precipitated with 30 mL of deionized water and washed twice with 30 mL of deionized water containing 10% (v / v) ethanol. The solid residue was freeze-dried overnight. Subsequently, the dried polymer was redissolved in DCM and purified by precipitation with 30 mL of ether. The supernatant is then discarded and the precipitate is vacuum dried overnight. The yellowish solid product obtained is the TDP-TXX drug conjugate. The dried TDP-TXX drug conjugate is stored in a desiccator for further use. The above TDP is: poly (triol dicarboxylic acid)-co-poly (ethylene glycol) polymer.

[0077] The samples prepared in the above examples and comparative examples were subjected to the following experiments.

[0078] Experiment 1

[0079] Step 1.1: The PTTX1 preparation mother solution with a concentration of 20 g / L (mg / mL) obtained in Example 1 is taken out from the refrigerator, and when it returns to room temperature, physiological saline for injection is added to prepare PTTX1 preparations with different concentrations (0.625 g / L to 16 g / L), and the preparations are refrigerated in the refrigerator for later use;

[0080] Step 1.2: The PTTX2 preparation mother solution with a concentration of 5 g / L obtained in Example 2 was taken out from the refrigerator, and after returning to room temperature, PTTX2 preparations with different concentrations were obtained by the same method as above.

[0081] Step 1.3: Accurately weigh 1.0 mg of TTX, add 100-200 μL of 0.6% acetic acid aqueous solution to dissolve it, then add normal saline for injection, dilute to 10 ml, and obtain a TTX preparation stock solution with a concentration of 0.1 g / L, divide it into 1 ml / tube, and store it at -20°C for later use;

[0082] Take out the 0.1 g / L TTX preparation mother solution from the refrigerator, and when it returns to room temperature, add normal saline for injection to prepare TTX preparations of different concentrations (0.125 mg / L ~ 4 mg / L), and refrigerate for later use.

[0083] Step 1.4: Experimental animals used mice, weighing 20g±2g, half male and half female. The experiment began after the mice were adaptively raised for 4 to 7 days. They were fasted for 12 hours before the experiment and had free access to water. After fasting for 12 hours, the mice were randomly divided into groups, with 10 mice in each group.

[0084] Step 1.5: After fasting for 12 hours, take out a group (10 mice) and inject about 40μL of the 16.0 g / L PTTX1 preparation prepared in the previous step into the thigh muscle of the mice at an intramuscular injection dose of 2μL / g body weight. No leakage should occur during the injection process. After the injection, observe for 20-30 minutes to determine the survival of the mice. If the number of dead mice exceeds half, reduce the concentration and repeat the experiment. If the number of dead mice is less than half, increase the concentration and repeat the experiment until the concentration of PTTX1 preparation with a lethality of 50% (LD50) is determined.

[0085] Step 1.6: Take out a group of 10 mice after fasting for 12 hours, and inject about 40μL of the 5.0g / L PTTX2 preparation prepared in the previous step into the thigh muscle of the mice according to the intramuscular injection dose of 2μL / g body weight. No leakage should occur during the injection process. Observe for 20-30 minutes after the injection to determine the survival of the mice. If the number of dead mice exceeds half, reduce the concentration and repeat the experiment. If the number of dead mice is less than half, increase the concentration and repeat the experiment until the concentration of PTTX2 preparation with a lethality of 50% (LD50) is determined.

[0086] Step 1.7: After fasting for 12 hours, take out a group (10 mice) and inject about 40μL of the 4 mg / L TTX preparation prepared in the previous step into the thigh muscle of the mice at an intramuscular injection dose of 2μL / g body weight. No leakage should occur during the injection process. Observe for 20-30 minutes to determine the survival of the mice. If the number of dead mice exceeds half, the concentration needs to be reduced and the experiment needs to be repeated. If the number of dead mice is less than half, the concentration needs to be increased and the experiment needs to be repeated until the concentration of TTX preparation with a lethality of 50% (LD50) is determined.

[0087] Refer to steps 1.1-1.7, take out a group (10 mice) of mice that have been fasted for 12 hours, and inject about 40μL of TDP-TTX preparation into the thigh muscle of the mice according to the intramuscular injection dose of 2μL / g body weight. No leakage should occur during the injection process. Observe for 20-30 minutes to determine the survival of the mice. If the number of dead mice exceeds half, the concentration needs to be reduced and the experiment needs to be repeated. If the number of dead mice is less than half, the concentration needs to be increased and the experiment needs to be repeated until the concentration of TDP-TTX preparation with a lethality of 50% (LD50) is determined.

[0088] The experimental results are shown in Table 1 below.

[0089]

[0090] The test showed that the LD50 concentrations of PTTX1, PTTX2 and TDP-TTX were much higher than 4.0 mg / L of TTX, indicating that the safety of PTTX1, PTTX2 and TDP-TTX was much higher than that of TTX.

[0091] Experiment 2

[0092] Step 2.1: The preparation of drugs with different concentrations (PTTX1 preparation, PTTX2 preparation, TTX preparation, TDP-TTX preparation) was the same as in Experiment 1. The normal saline injection was used as the blank control group.

[0093] Step 2.2: Experimental animals used mice, weighing 20g±2g, half male and half female. The experiment began after the mice were adaptively raised for 4 to 7 days. They were fasted for 12 hours before the experiment and had free access to water. After fasting for 12 hours, the mice were randomly divided into groups, with 10 mice in each group.

[0094] Step 2.3: After fasting for 12 hours, take out one group (10 mice) and inject different concentrations of drugs (injection dose is about 40μL) at an intramuscular injection dose of 2μL / g body weight, and inject normal saline at 2μL / g body weight in the blank control group. No leakage should occur during the injection process. Start the timer after the injection of drugs or normal saline is completed, and inject 0.6% acetic acid aqueous solution into the mouse peritoneum 40 minutes later.

[0095] Step 2.4: After the injection of acetic acid aqueous solution, place the mice separately, observe and record the number of twisting of each mouse within 15 minutes, and the twisting reaction is positive when the abdomen is concave, the trunk and hind limbs are stretched, and the buttocks are raised. According to the average number of twisting of each group of mice, calculate the pain inhibition rate of the preparations of different concentrations corresponding to the group, and the calculation formula is:

[0096] Pain inhibition rate (%) = (average number of writhings in the blank control group - average number of writhings in the drug-treated group) / average number of writhings in the blank control group × 100%.

[0097] The different concentrations of the preparations that inhibited pain by 50% (EC50) were determined by increasing or decreasing the concentrations. The results are shown in Tables 2 and 3.

[0098]

[0099]

[0100] The data in Tables 2 and 3 show that PTTX1, PTTX2, and TDP-TTX have analgesic effects comparable to those of TTX, and compared with Table 1, the drug windows of PTTX1, PTTX2, and TDP-TTX are significantly higher than those of TTX. However, the concentration of TDP-TTX used is 3.12 times that of PTTX1. That is, the effective ingredient TTX loaded in TDP-TTX is too low (0.1-1.6 μg / mg in the literature), and TDP is synthesized from polyethylene glycol (PEG), sebacic acid, and glycerol as raw materials. According to previous literature reports, the use of PEGylated drugs to treat patients who have acquired anti-PEG antibodies will lead to accelerated blood clearance, reduced drug efficacy, hypersensitivity reactions, and in some cases, life-threatening side effects.

[0101] Experiment 3

[0102] In vitro release experiment process of PTTX1 and PTTX2: Accurately weigh 1.3 mg of PTTX1 prepared in Example 1, dissolve it in 130 microliters of PBS (pH=7.4), and prepare a solution with a concentration of 10 g / L. At the same time, weigh 1.0 mg of PTTX2 prepared in Example 2, dissolve it in 100 microliters of PBS (pH=7.4), and prepare a solution with a concentration of 10 g / L. Take 100 μL of each solution and add it to the dialysis bag (molar mass: 1000Da) and seal it. Place the dialysis bag in a sample bottle containing 10 mL PBS (pH=7.4) and tighten the bottle cap. Then put the sample bottle into a constant temperature incubation shaker (37°C, 90rpm). Take samples at 0.5h, 1h, 3h, 6h, 9h, 12h, 24h, 2d, 3d, 5d, and 7d respectively. After sampling, use TTX Elisa kit to test the release amount. The results are as follows Figure 7 shown.

[0103] Figure 7 Comparing the in vitro release curve experimental results of PTTX1 and PTTX2 in the literature with those of TDP-TTX in the reference, Figure 7 In A, PTTX1 can release 429.0 ng of TTX at 72 h and 543.65 ng of TTX at 168 h. Figure 7 In B, PTTX2 can release 240.9 ng of TTX at 72 h and 248.7 ng of TTX at 168 h. Figure 7 C can be estimated (according to the literature reports, the synthesized T g D8P 200-TTX conjugate has a TTX loading of 0.31 μg / mg) TDP-TTX can release about 46.5 ng of TTX in 72h and about 86.8 ng of TTX in 168h. Due to the small amount of TDP-TTX released, the literature shows that injecting TDP-TTX into the sciatic nerve of mice can effectively anestheticize it. However, if it is used for systemic administration, it may not reach the effective dose, that is, it cannot have an anesthetic effect.

[0104] Experiment 4

[0105] 4.1 Cell culture

[0106] According to previous studies, cell culture of C2C12 mouse myoblasts (Punosai, Wuhan) and PC12 rat adrenal pheochromocytoma cells (ATCC, CRL-1722) (Punosai, Wuhan) was performed.

[0107] C2C12 cells were cultured in DMEM (bkmam) with 20% FBS and 1% penicillin-streptomycin (Shanghai Yuanye Biotechnology Co., Ltd.). The cells were seeded at 50,000 cells / ml on a 24-well plate and incubated for 10-14 days in DMEM with 2% horse serum (Wuhan Punosai) and 1% penicillin-streptomycin to differentiate into myotubules.

[0108] PC12 cells were grown in DMEM with 12.5% ​​horse serum, 2.5% FBS, and 1% penicillin-streptomycin. Cells were seeded onto 24-well plates, and 50 ng / mL nerve growth factor (Punosai, Wuhan) was added 24 hours after seeding.

[0109] 4.2 Cell viability

[0110] The cells (1x10 4 / well) were incubated with various concentrations of carboxylated dextran and various concentrations of carboxylated β-cyclodextrin for 24 hours. After incubation, the cells were washed 5 times with PBS to remove the polymer, and the cell viability was determined by MTT. In short, the culture supernatant from the control wells and the polymer-containing wells was collected, and the cells were incubated with MTT (0.5 mg / mL, 3 h). The formation was dissolved in 200 μL DMSO and the optical density was measured at 550 nm. The absorbance of the control well was assumed to be 100%, and the cell viability of the treated wells relative to the control wells was determined.

[0111] 4.3 Cell viability test results

[0112] like Figure 8 and 9As shown, there was no significant difference in the ratio of cell viability to blank control cell viability at different carboxylated dextran concentrations, indicating that the polymer carboxylated dextran would not reduce cell activity and had good cell compatibility. Fig.10 The live cells were shown to be green, further demonstrating that the carboxylated dextran had little toxicity.

[0113] like Fig.11 and 12 As shown, there is no significant difference in the ratio of cell viability to blank control cell viability at different carboxylated β-cyclodextrin concentrations, indicating that the polymer carboxylated β-cyclodextrin does not reduce cell activity and has good cell compatibility. Fig.13 The live cells were shown to be green, further demonstrating that carboxylated β-cyclodextrin had little toxicity.

[0114] Experiment 5

[0115] 5.1 Establishment of rat skin cancer pain model:

[0116] (1) Experimental animals

[0117] Healthy adult female (SD) rats, weighing 140-150 g, were purchased from Jiangsu Wukong Pharmaceutical Co., Ltd. and were kept in separate cages with adequate food and water. The laboratory was kept at a constant temperature and humidity, and the rats were allowed to eat and drink freely. This experiment strictly complied with the requirements of the International Pain Society for the use of awake animals in pain experimental research. During the experiment, laboratory operation specifications were also strictly followed.

[0118] (2) Experimental cells

[0119] MADB106 rat breast cancer cell line was purchased from Shanghai Yuhao Biotechnology Co., Ltd.

[0120] (3) Preparation and grouping of rat skin cancer pain model

[0121] Ten rats were randomly divided into a normal control group (hereinafter referred to as the "control group") and a tumor cell inoculation model group (hereinafter referred to as the "model group"), with 5 rats in each group. The model group rats were first anesthetized by intraperitoneal injection of 10% chloral hydrate; the rat foot pad skin was disinfected with iodine tincture, and 100 uL of MADB106 breast cancer cell suspension was slowly injected into the subcutaneous part of the foot pad of the model group rats using a 1 mL syringe, and then iodine tincture was used for local pressing for 1 min. After the experimental operation was completed, the rats were returned to their original cages after waking up; the rats were then closely observed for adverse reactions.

[0122] like Fig.14As shown in the figure, the soles of the feet of the rats in the model group began to show different degrees of redness and swelling from 3 days after inoculation, and the redness and swelling became more obvious as the experiment progressed. This is because tumor cells have always existed and continued to grow. If the redness and swelling were caused by inflammation, the soles of the rats' feet would also be red and swollen at first, but as time went on, the redness and swelling subsided on the 10th day.

[0123] 5.2 Mechanical pain detection:

[0124] The mechanical paw withdrawal threshold (MPWT) of the hind paw of the operated side was compared among the rats in each group 20 days after modeling. The mechanical paw withdrawal threshold of the model group was lower. There was a significant statistical difference between the model group and the control group ( P <0.05).

[0125]

[0126] 5.3 Expression of inflammatory factors:

[0127] The skin tissues of the control group and the model group were taken for cytokine concentration determination at 20 days. The concentrations of IL-1β and TNF-a were detected by IL-1β ELISA kit (Lianke Biotechnology) and TNF-a ELISA kit (Lianke Biotechnology). The experimental steps were carried out according to the instructions.

[0128] The experimental results are as follows: after rats were inoculated with tumor cells, the levels of IL-1β and TNF-а in the tumor-bearing skin tissue were higher than those in the blank control group (both P <0.05).

[0129]

[0130] Studies have shown that NGF, brain-derived nerve growth factor (BDNF), neurotrophin-3 (NT-3), IL-1β, TNF-a, interleukin (I-6) and transforming growth factor β (TGF-β) are all involved in the formation of cancer pain (Wacnik P, Eikmeier L, Ruggles T, et al. Functional interactions between tumor and peripheral nerve: morphology, algogen identification, and behavioral characterization of a new murine model of cancer pain. J Neurosci, 2002, 7(2)9367~9376.). IL-1 is mainly produced by macrophages. Under normal circumstances, only some tissues have a small amount of IL-1. Only when subjected to certain exogenous stimuli can most cells synthesize and secrete IL-1. IL-1 exists in the form of IL-1а and IL-1β. IL-1β is an important pro-inflammatory factor. Studies have confirmed (MH. van den Beuken-vanEverdingen, LM Hochstenbach, EA Joosten, VC TjanHeijnen, DJ Janssen, Update on prevalence of pain in patients with cancer: systematic review andmeta-analysis, Pain Symptom Manag. 5l (2016)1070-1090.) that IL-1β expression is significantly increased in the rat bone cancer pain model.

[0131] TNF-a is a pro-inflammatory cytokine that is mainly synthesized and secreted by macrophages and monocytes and then produced in an autocrine and paracrine manner. Experiments showing that TNF-a is produced more in many pathological conditions have found that TNF-a can not only activate osteoclasts to participate in bone destruction, but also play an important role in pain-sensitive behavior. Antagonizing TNF-a can not only reduce bone destruction, but also alleviate hyperalgesia. A study on the correlation between TNF-a and cancer pain found that (H. Dai, R. Li, T. Wheeler et al., “Enhanced survival in peri-neural invasion of pancreatic cancer: an in vitro approach," Human Pathology, vol. 38, no. 2, pp299-307, 2007.) it is also involved in the occurrence and development of cancer pain.

[0132] In this experiment, in the tumor cell skin cancer pain model, we detected the concentration of cytokines (IL-1β, TNF-a) in rat skin tissue by enzyme-linked immunosorbent assay. We found that the concentrations of IL-1β and TNF-a were measured on the 20th day after tumor cell inoculation and compared with the concentrations of IL-1β and TNF-a in the control group on the 20th day after surgery. The model group was higher than the control group. Statistical comparison showed that the cytokine concentrations after tumor cell inoculation were higher than those in the control group. P <0.05, the difference between the two groups was statistically significant. The study showed that the expression of IL-1β and TNF-a in tumor-bearing skin tissue increased, indicating that inflammatory factors may participate in the hyperalgesia caused by tumor cells. Because the rat skin cancer pain model has a good simulation of the pathological process related to cancer pain, this model will help to clarify the mechanism of cancer pain and develop related measures.

[0133] 5.4 Mechanical pain sensitivity test after drug administration:

[0134] To investigate whether intraperitoneal injection of tetrodoxin conjugate can relieve mechanical hyperalgesia in cancer pain model and prolong drug release time. Tetrodoxin conjugate was injected intraperitoneally 20 days after modeling. Electronic Von Frey was used to measure plantar MPWT of rats 1 hour before administration, 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours and 12 hours after administration. Injection was performed according to the EC50 of PTTX1, PTTX2, TDP-TTX and TTX. Rats were placed in a plexiglass box with a hollow metal grid at the bottom. After acclimating to the environment for 20 minutes, the measurement was started when the rats were in a resting state. Von Frey filaments stimulated the plantar skin of rats from bottom to top. When a rapid paw retraction reaction occurred, the stimulation stopped automatically, and the pressure value was recorded. The measurement was performed 5 times, with an interval of 3 minutes each time, and the average value was taken. After the experiment, the rats were returned to the cage for continued feeding. The rats were in good condition and no deaths occurred during 30 days of feeding.

[0135] As shown in Table 6, intraperitoneal injection of TTX can significantly increase the MPWT of model rats, and the analgesic effect starts at 0.5h after administration and the efficacy reaches a peak, and the efficacy disappears at 4h. Injection of TDP-TTX has a certain analgesic effect, but the efficacy is not significant. Intraperitoneal injection of PTTX2 (carboxylated β-cyclodextrin-TTX) has an analgesic effect at 0.5h after administration, reaches a peak efficacy at 4h, and the analgesic duration is as long as 12h. Intraperitoneal injection of PTTX1 (carboxylated dextran-TTX) has an analgesic effect at 0.5h after administration, reaches a peak efficacy at 2h, and the analgesic duration is as long as 12h, and it still has an analgesic effect. It is reasonable to speculate that PTTX1 is still continuing analgesia. The analgesic time of PTTX1 and PTTX2 is longer than that of TTX, and the analgesic efficacy is much higher than that of TDP-TTX.

[0136]

[0137] In summary, the present application provides artificially synthesized high molecular polymer-TTX conjugates PTTX1 (carboxylated dextran-TTX) and PTTX2 (carboxylated β-cyclodextrin-TTX). This polymer can significantly improve the systemic analgesic therapeutic index, and the hydrolysis product (carboxylated dextran or carboxylated β-cyclodextrin) has no cytotoxicity.

[0138] Studies have shown that PTTX1 and PTTX2 can significantly expand the drug treatment window of TTX, and in the cancer pain model, all PTTX can significantly extend the analgesic sustained release time, among which PTTX1 has an analgesic time of more than 3 times that of TTX, and has no systemic toxicity. This is because PTTX is covalently linked to the biodegradable and biocompatible carboxylated glucan backbone or carboxylated β-cyclodextrin backbone by TTX through hydrolyzable ester bonds, and the hydrolyzable ester bonds control the continuous release of TTX in its natural bioactive form and avoid burst release.

[0139] TTX has a very narrow therapeutic window, which requires controlling the release rate of TTX to avoid systemic toxicity while prolonging the efficacy. PTTX can precisely control the hydrolysis rate of the ester bond to keep the drug level within the therapeutic window and release TTX higher than the effective dose. For example, PTTX1 can release 543.65 ng of TTX in 168h, while TDP-TTX in the literature can only release about 86.8 ng of TTX in 168h. That is, the release amount of TDP-TTX is low, and it can achieve the corresponding efficacy when used for local administration, but if it is used for systemic administration, it may have no direct pharmacological effect.

[0140] Animal experiments have shown that both PTTX and TDP-TTX significantly broaden the drug window of TTX. The EC50 of TDP-TTX is 3.13 times that of PTTX1 and 25 times that of PTTX2, which means that the dosage of TDP-TTX needs to be more than 3 times that of PTTX1 or 25 times that of PTTX2 to reach the effective dose. In addition, PTTX is highly soluble in water and PBS, while TDP-TTX itself has very poor solubility and is difficult to reach an effective dose. It needs to be in PEG to form an injectable preparation. According to previous literature reports, the use of PEGylated drugs to treat patients who have acquired anti-PEG antibodies will lead to accelerated blood clearance, reduced drug efficacy, hypersensitivity reactions, and in some cases, life-threatening side effects.

[0141] In addition, in the cancer pain model experiment, the present application conducted an analgesic experiment in rats by systemic administration (intraperitoneal injection) to verify the analgesic effect of PTTX and evaluate the safety of PTTX. The experimental results showed that PTTX significantly reduced the pain of skin cancer rats induced by MADB106 breast cancer tumor cells. The analgesic duration was up to 12 hours and even longer. No animals died during the experiment and no signs of acute poisoning were found. TDP-TTX in the literature was injected locally into the sciatic nerve of experimental mice to exert its pharmacological effect. In this experiment, TDP-TTX was also administered by intraperitoneal injection, and the analgesic effect was not significant and the efficacy was much lower than that of PTTX. Therefore, when TDP-TTX is administered systemically, the analgesic effect cannot be obtained and the safety needs to be considered.

Claims

1. A tetrodoxin conjugate, characterized in that: The structural formula of the tetrodoxin conjugate is selected from formula (I) or (II): (I), where n=1-20000; (II), wherein R=COCH2CH2COOH or H.

2. The method for preparing the tetrodoxin conjugate according to claim 1, characterized in that: The tetrodoxin conjugate is obtained by taking an artificially synthesized carboxyl-containing polymer compound and tetrodoxin as raw materials, adding a catalyst and a solvent, and then performing a coupling reaction.

3. The preparation method according to claim 2, characterized in that: The catalyst is selected from any one of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, 1-ethyl-(3-dimethylaminopropyl) carbodiimide, N,N'-dicyclohexylcarbodiimide, and N,N-dimethylaminopyridine or other organic bases; the solvent for the coupling reaction is selected from a non-hydroxyl solvent.

4. The preparation method according to claim 3, characterized in that: The solvent for the coupling reaction is selected from any one of dimethyl sulfoxide and N,N-dimethylformamide.

5. Use of the tetrodotoxin conjugate according to claim 1 in the preparation of a medicament for treating neuropathic pain caused by chemotherapy.

6. A tetrodoxin conjugate preparation, characterized in that: The tetrodoxin conjugate preparation is prepared by adding pharmaceutically acceptable excipients to the tetrodoxin conjugate according to claim 1.

7. The tetrodoxin conjugate preparation according to claim 6, characterized in that: The pharmaceutically acceptable excipients include inert organic carriers and / or inorganic carriers.

8. The tetrodoxin conjugate preparation according to claim 6, characterized in that: The dosage form of the tetrodotoxin conjugate preparation is injection.

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