Tetrodotoxin conjugate as well as preparation method and application thereof

Abstract

The invention discloses a tetrodotoxin conjugate as well as a preparation method and application thereof. The preparation method comprises the following steps: by taking a carboxyl-containing high-molecular compound and tetrodotoxin as raw materials, adding a catalyst and a solvent, and carrying out coupling reaction to obtain the tetrodotoxin conjugate. The carboxyl-containing high-molecular compound is selected from alginic acid or polyacrylic acid. The tetrodotoxin conjugate can be applied to preparation of sodium channel blockers and drugs for treating neuropathic pain caused by chemotherapy. The tetrodotoxin conjugate disclosed by the invention has an analgesic curative effect associated with TTX, a drug window is enlarged, and the medication safety 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), also known as Halneuron, Tectin or Tetrodin, is an alkaloid found in pufferfish (commonly known as pufferfish) and other organisms. It is an aminoperhydroquinazoline compound and one of the most toxic neurotoxins found in nature. It was once considered the most toxic non-protein toxin in nature. Its molecular formula is C 11 H 17 O8N3, molecular weight is 319.27, CAS: 4368-28-9, its molecular structure is as follows:

[0003] In 1909, Japanese scientist Yoshizumi Tahara discovered and isolated tetrodotoxin for the first time; in 1955, Hirata Yoshimasa successfully separated tetrodotoxin from puffer fish, and then artificially synthesized tetrodotoxin in 1972.

[0004] Tetrodoxin is a typical sodium channel blocker, and its mechanism of action includes Nav1.7 blocker, Nav1.8 blocker and Nav1.5 blocker. The mechanism of action can be simply described as follows: 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 the animal's nerve excitation and conduction, the regulatory function of the central nervous system, and a series of physiological functions such as heart beat, smooth muscle peristalsis, skeletal muscle contraction, hormone secretion, etc., leading to muscle and nerve paralysis.

[0005] Compared with commonly used anesthetics, the anesthetic effect of tetrodoxin is more than 10,000 times stronger and lasts longer, and can completely block the withdrawal symptoms induced by the opioid natron after morphine withdrawal. Tetrodoxin can exert a strong analgesic and anesthetic effect in a trace amount in the body. However, the disadvantage of tetrodoxin is that it has poor solubility, is slightly soluble in weak acids, anhydrous ethanol, and ether, and is almost insoluble in other organic reagents. It is also highly active, highly toxic, and has a low therapeutic index, which seriously affects its clinical research and application. Among them, the safety and stability of tetrodoxin has always been the main obstacle that is difficult to solve. It is speculated that the reason why the analgesic effect of monomer TTX is unstable is that it is impossible to determine an effective safe dose. When the dose is too large, a part of the TTX molecules pass through the blood-brain barrier to produce a certain analgesic effect, but because of its high toxicity, it can cause severe hypotension and respiratory depression, so safety cannot be guaranteed; and when the dose is too small, the TTX molecules will be completely blocked by the blood-brain barrier, and thus no analgesic effect can be produced (as described in reference 1).

[0006] Those skilled in the art continue to develop tetrodotoxin derivatives, but it is clear that various technical routes have their own disadvantages, such as high burst drug release, low mechanical strength of the gel leading to potential dose dumping, poor biocompatibility of the polymer micelle system and gradual decrease in the pH value of the system due to acidic degradation, and potential safety hazards to the human body, environment and organisms caused by irritating small molecule acyl chloride raw materials (as described in References 2, 3, 4 and 5).

[0007] The potential application areas of tetrodoxin are very wide, including nervous system diseases, cancer pain, neuralgia, anesthesia, heroin dependence, etc. However, the current application direction is mainly to relieve the pain of cancer patients. It can be seen that the problems existing in the use of tetrodoxin in the prior art have not been properly solved, which limits the application of tetrodoxin.

[0008] 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.

[0009] Literature 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.

[0010] Document 3: Schmaljohann D. Thermo-and pH-responsive polymers in drug delivery[J]. Advanced drug delivery reviews, 2006, 58(15): 1655-1670.

[0011] 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.

[0012] 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

[0013] 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:

[0014] ; wherein R is the skeleton of a carboxyl-containing polymer compound, and the carboxyl-containing polymer compound is selected from alginic acid or polyacrylic acid.

[0015] Preferably, the structural formula of the tetrodoxin conjugate is selected from formula (I) or (II): (I), in formula (I), m=1-2000, n=1-1900; (II), wherein m=1-9000, n=1-10000.

[0016] The tetrodoxin conjugate of formula (I) is hereinafter referred to as PTTX1; the tetrodoxin conjugate of formula (II) is hereinafter referred to as PTTX2.

[0017] The second object of the present invention is to provide a method for preparing the tetrodoxin conjugate, which comprises taking a carboxyl-containing polymer compound and tetrodoxin as raw materials, adding a catalyst and a solvent, and obtaining the tetrodoxin conjugate through a coupling reaction. The carboxyl-containing polymer compound is selected from alginic acid or polyacrylic acid.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] Preferably, the dosage form of the tetrodotoxin conjugate preparation is an injection.

[0024] Compared with the prior art, the present invention has the following advantages: 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; in addition, the natural polymer has good biocompatibility and is degradable, and will not cause harm to human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the general formula of PTTX of the present invention.

[0026] Figure 2 is the H NMR spectrum of alginate in D2O, 1 H NMR (400 MHZ, D2O) δ: 3.5~5.2 ppm.

[0027] Figure 3 This is the H NMR spectrum of TTX in 4%CD3COOD / 96%D2O. 1H 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).

[0028] Figure 4 This is the H NMR spectrum of PTTX1 in D2O.

[0029] Figure 5 This is the H NMR spectrum of PTTX2 in D2O.

[0030] Figure 6 The morphological changes of rats after inoculation of MADB106 cells in the rat plantar.

[0031] Figure 7 Cell viability of PC12 cells incubated in different concentrations of alginate 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 alginate (w / w).

[0032] Figure 8 Cell viability of C2C12 cells incubated in different concentrations of alginate for 24 hours, where the ordinate represents cell viability (100%) and the abscissa represents the concentration of alginate (w / w).

[0033] Fig. 9 This is a living color chromosome-alginate map.

[0034] Fig.10 The cell survival rate of PC12 cells incubated with different concentrations of polyacrylic acid for 24 hours, where the ordinate represents the survival rate (100%) and the abscissa represents the concentration of polyacrylic acid (w / w).

[0035] Fig.11 The cell survival rate of C2C12 cells incubated in different concentrations of polyacrylic acid for 24 hours, where the ordinate represents the survival rate (100%) and the abscissa represents the concentration of polyacrylic acid (w / w).

[0036] Fig.12 For the living color chromosome-polyacrylic acid map.

[0037] Fig.13 In vitro release curves of different TTX conjugates, where: Fig.13 A is the in vitro release curve of PTTX1 in PBS (pH = 7.4); Fig.13B is the in vitro release curve of PTTX2 in PBS (pH = 7.4); Fig.13 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.). DETAILED DESCRIPTION

[0038] 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.

[0039] 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. Alginate and polyacrylic acid were purchased from Myrel, dimethyl sulfoxide, N,N-dimethylaminopyridine, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were purchased from Anergy, tetrodoxin was from Huamu, and TTX Elisakit was from Reagen. The abbreviations used in the description of this application are as follows: Dimethyl sulfoxide: DMSO, N,N-dimethylaminopyridine: DMAP, 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride: EDC . HCl, Tetrodotoxin: TTX, Tetrodoxin conjugate: PTTX (its general structural formula is as follows Figure 1 as shown).

[0040] 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).

[0041] Example 1

[0042] Step 1, preparing PTTX-1 (PTTX1), the synthetic route is as follows:

[0043] In a nitrogen atmosphere, TTX (0.6 g) and EDC were dissolved and dispersed in DMSO (1.5 L) in a reaction bottle. . HCl (0.76 g), DMAP (46 mg) and alginic acid (8.9 g, its H NMR spectrum is as follows Figure 2 The reaction mixture was dialyzed against water for 24 h using a dialysis bag (Mw: 1000 Da) and freeze-dried (-40 ° C, 72 h) to obtain a white solid PTTX1, which weighed 8.10 g after washing and drying, and the reaction yield was greater than 85%. Figure 4 The H NMR spectrum of PTTX1 in D2O shows that the peaks of TTX and alginate overlap when the chemical shift is lower than 5.2 ppm. TTX can be observed at 5.45 ppm (its H NMR spectrum is shown in Figure 2). Figure 3 The characteristic peaks are shown in Figure 2.

[0044] Step 2: Preparation of Tetradoxine Conjugate-1 (PTTX1) Preparation Stock Solution 5 mg of PTTX1 prepared in step 1 of this example was accurately weighed and then dissolved in physiological saline to a fixed concentration of 5000 mg / L (5 g / L) to obtain a PTTX1 preparation mother solution, which was stored at -20°C for future use.

[0045] Example 2 Step 1, prepare PTTX2 conjugate-2 (PTTX2), the synthetic route is as follows:

[0046] In a nitrogen atmosphere, TTX (0.6 g) and EDC were dissolved and dispersed in DMSO (1.5 L) in a reaction bottle. . HCl (0.76 g), DMAP (46 mg) and polyacrylic acid (1 g) were reacted at room temperature for 5 to 7 days. After the reaction, the reaction solution was dialyzed against water using a dialysis bag (Mw: 1000 Da) for 24 hours. After the dialysis, it was freeze-dried (-40°C, 72 hours). The freeze-dried product was a white solid PTTX2, which weighed 1.50 grams after rinsing and drying, and the reaction yield was greater than 90%. Figure 5 This is the hydrogen nuclear magnetic resonance spectrum of PTTX2 in D2O. The characteristic peak of TTX can be observed at 5.45 ppm.

[0047] Step 2: Preparation of Tetradoxine Conjugate-2 (PTTX2) Preparation Stock Solution 5 mg of PTTX2 prepared in step 1 of this example was accurately weighed and then dissolved in physiological saline to a fixed concentration of 5000 mg / L (5 g / L) to obtain a PTTX2 preparation mother solution, which was stored at -20°C for future use.

[0048] Comparative Example 1 Synthesis of TDP-TXX drug conjugates: 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 by rotary evaporation. 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.

[0049] The samples prepared in the above examples and comparative examples were subjected to the following experiments.

[0050] Experiment 1 Step 1.1: The PTTX1 preparation mother solution with a concentration of 5000 mg / L obtained in Example 1 was taken out from the refrigerator, and when it returned to room temperature, physiological saline for injection was added to prepare PTTX1 preparations with different concentrations (1250 mg / L~39.0625 mg / L), and the preparations were refrigerated in the refrigerator for later use; Step 1.2: Accurately weigh 1 mg of TTX, dissolve it in 100-200 μL of 0.6% acetic acid aqueous solution, add normal saline for injection, and dilute to 10 mL to obtain a TTX preparation mother solution with a concentration of 100 mg / L, and store it at -20°C for later use; Take out the 100 mg / 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 (6 mg / L~0.125 mg / L), and refrigerate for later use.

[0051] Step 1.3: 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.

[0052] Step 1.4: After fasting for 12 hours, take out a group (10 mice) and inject about 40μL of the 1250mg / 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. 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 PTTX1 preparation with a lethality of 50% (LD50) is determined.

[0053] Step 1.5: Take out a group of 10 mice after fasting for 12 hours, and inject about 40μL of the 6mg / L TTX 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 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.

[0054] Refer to steps 1.1-1.5, take out a group (10 mice) of mice after fasting for 12 hours, and inject about 40μL of TDP-TTX preparation into the thigh muscle of 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.

[0055] The experimental results are shown in Table 1.

[0056] The above test shows that the LD50 concentration of PTTX1 preparation is 206 mg / L, and the LD50 concentration of TDP-TTX preparation is 19400 mg / L, which is much higher than the 4 mg / L of TTX preparation. This shows that the safety of PTTX1 preparation and TDP-TTX preparation is much higher than that of TTX preparation.

[0057] Experiment 2 Step 2.1: Preparation of drugs of different concentrations (PTTX1 preparation, TTX preparation, TDP-TTX preparation) was the same as in Experiment 1. The normal saline injection was used as the blank control group.

[0058] 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.

[0059] 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.

[0060] 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: 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%.

[0061] 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.

[0062]

[0063]

[0064] The data in Tables 2 and 3 show that PTTX1 and TDP-TTX have analgesic effects comparable to those of TTX, and compared with Table 1, the drug windows of PTTX1 and TDP-TTX are significantly higher than those of TTX. However, the data show that the LD50 of TDP-TTX is 94 times that of PTTX1, and the EC50 of TDP-TTX is 40 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.

[0065] Experiment 3 Referring to Experiment 1, the mother solution of PTTX2 preparation with a concentration of 5000 mg / L was added with injection saline to prepare PTTX2 preparations with concentrations of 1250 mg / L to 39.0625 mg / L. The PTTX2 preparations were used for toxicity tests. The results are shown in Table 4.

[0066]

[0067] The above test shows that the LD50 concentration of PTTX2 preparation is 500 mg / L, which is much higher than 4 mg / L of TTX preparation. This shows that the safety of PTTX2 preparation is much higher than that of TTX preparation.

[0068] Experiment 4 Referring to Experiment 2, the preparation of drugs (PTTX2 preparations) of different concentrations was the same as Experiment 3. The normal saline injection was used as the blank control group. The results of the acetic acid writhing test in mice are shown in Tables 5 and 6.

[0069]

[0070] The data in Table 5 above indicate that PTTX2 has analgesic efficacy associated with TTX, and the drug window is enlarged.

[0071]

[0072] The data in Table 6 above show that the PTTX2 preparation has analgesic effect.

[0073] Experiment 5 In vitro release experiment process of PTTX1 and PTTX2: Accurately weigh 1.0 mg of PTTX1 prepared in Example 1, dissolve it in 100 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 incubator 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 Fig.13 shown.

[0074] Fig.13 Comparing the in vitro release curve experimental results of PTTX1 and PTTX2 in the literature with those of TDP-TTX in the references, Fig.13In A, PTTX1 can release 446.5 ng of TTX at 72 h and 482.2 ng of TTX at 168 h. Fig.13 PTTX2 in B can release 313.9 ng of TTX at 72 h and 408.7 ng of TTX at 168 h. Fig.13 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.

[0075] Experiment 6 6.1 Establishment of rat skin cancer pain model: (1) Experimental animals 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.

[0076] (2) Experimental cells MADB106 rat breast cancer cell line was purchased from Shanghai Yuhao Biotechnology Co., Ltd.

[0077] (3) Preparation and grouping of rat skin cancer pain model 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 with 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 the original cage after waking up and continued to be raised; the rats were then closely observed for adverse reactions.

[0078] like Figure 6 As 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.

[0079] 6.2 Mechanical pain detection: 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).

[0080]

[0081] 6.3 Expression of inflammatory factors: The skin tissues of the control group and the model group were taken at 20 days for the cytokine test. 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.

[0082] 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).

[0083]

[0084] 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-1a 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.

[0085] 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.

[0086] 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.

[0087] 6.4 Mechanical allodynia test after drug administration: 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, and 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 on the 20th day. 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.

[0088] As shown in Table 9, intraperitoneal injection of TTX can significantly increase the MPWT of model rats. 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 PTTX1 (alginate-TTX) has an analgesic effect at 0.5h after administration, reaches a peak efficacy at 1h, and then the efficacy decreases, reaching the second peak efficacy at 6h. The analgesic effect lasts for up to 12h, and still has a certain analgesic effect. Intraperitoneal injection of PTTX2 (polyacrylic acid-TTX) has an analgesic effect at 0.5h after administration, is in a relatively mild analgesic state, and disappears at 12h. 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.

[0089]

[0090] Experiment 7 7.1 Cell culture 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.

[0091] 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.

[0092] 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.

[0093] 7.2 Cell viability The cells (1x10 4 / hole) were incubated with various concentrations of alginic acid and various concentrations of polyacrylic acid 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. It was assumed that the absorbance of the control well was 100%, and the cell viability of the treated wells relative to the control wells was determined.

[0094] 7.3 Cell viability test results like Figure 7 and 8 As shown, there is no significant difference in the ratio of cell viability to blank control cell viability at different alginate concentrations, which indicates that alginate does not reduce cell activity and has good cell compatibility. Fig. 9 The living cells were shown to be green, further proving that alginate was not toxic to the cells.

[0095] like Fig.10 and 11 As shown, there was no significant difference in the ratio of cell viability to blank control cell viability at different polyacrylic acid concentrations, indicating that polyacrylic acid would not reduce cell activity and had good cell compatibility. Fig.12 The living cells were shown to be green, further proving that polyacrylic acid had little toxicity to cells.

[0096] In summary, the present application provides natural high molecular polymer-TTX conjugates PTTX1 (alginate-TTX) and PTTX2 (polyacrylic acid-TTX), which can significantly improve the systemic analgesic therapeutic index, and the hydrolysis products (alginate or polyacrylic acid) are non-cytotoxic.

[0097] 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, which is more than 3 times that of TTX, without systemic toxicity. This is because PTTX is covalently linked to the biodegradable and biocompatible trehalose or polyacrylic acid 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.

[0098] 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 482.2 ng of TTX in 168h, and PTTX2 can release 408.7ng of TTX in 168h. In contrast, 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.

[0099] Animal experiments have shown that both PTTX and TDP-TTX significantly broaden the drug window of TTX. The EC50 of TDP-TTX is 40 times that of PTTX1 and 47.67 times that of PTTX2, which means that the dosage of TDP-TTX needs to be more than 40 times that of PTTX1 or 47.67 times that of PTTX2 to reach the effective dose, which means that the dosage is too much. In addition, PTTX is very soluble in water and PBS, while TDP-TTX itself has very poor solubility and it 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.

[0100] 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 general formula of the tetrodoxin conjugate is: ; wherein R is the skeleton of a carboxyl-containing polymer compound, and the carboxyl-containing polymer compound is selected from alginic acid or polyacrylic acid.

2. The tetrodoxin conjugate according to claim 1, characterized in that: The structural formula of the tetrodoxin conjugate is selected from formula (I) or (II): (I), in formula (I), m = 1-2000, n = 1-1900; (II), wherein m=1-9000, n=1-10000.

3. The method for preparing the tetrodoxin conjugate according to any one of claims 1 to 2, characterized in that: The carboxyl-containing polymer compound and tetrodoxin are used as raw materials, a catalyst and a solvent are added, and the tetrodoxin conjugate is obtained through a coupling reaction.

4. The preparation method according to claim 3, 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.

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

6. Use of the tetrodotoxin conjugate according to any one of claims 1 to 2 in the preparation of a sodium channel blocker and a drug for treating neuropathic pain caused by chemotherapy.

7. A tetrodoxin conjugate preparation, characterized in that: The tetrodoxin conjugate preparation is prepared by adding pharmaceutically acceptable excipients to the tetrodoxin conjugate according to any one of claims 1 to 2.

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

9. The tetrodoxin conjugate preparation according to claim 7, characterized in that: The dosage form of the tetrodotoxin conjugate preparation is an injection.

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