Tetrodotoxin conjugate, preparation method, and use

Abstract

Provided are a tetrodotoxin conjugate, a method for preparing same, and use thereof. A carboxyl-containing macromolecular compound and tetrodotoxin are used as the starting materials, and after a catalyst and a solvent are added, the tetrodotoxin conjugate is obtained by means of a coupling reaction. The carboxyl-containing macromolecular compound is selected from carboxylated dextran, carboxylated β-cyclodextrin, alginic acid, and polyacrylic acid. The tetrodotoxin conjugate can be used in the preparation of a sodium channel blocker and a medicament for treating neuropathic pain caused by chemotherapy, and possesses an analgesic effect associated with tetrodotoxin (TTX). In addition, the therapeutic window is increased, thereby greatly improving the safety.

Description

Titridoxin conjugates, preparation methods and applications Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically to tetrodoxin conjugates, methods for preparing the tetrodoxin conjugates, and applications of the tetrodoxin conjugates. Background Technology

[0002] Tetrodotoxin (TTX), also known as Halneuron, Tectin, or Tetrodin, is an alkaloid found in pufferfish and other organisms. It is an amino-perhydroquinazoline compound and is one of the most potent neurotoxins found in nature, once considered the most potent non-protein toxin in nature. Its molecular formula is C2. 11 H 17 O8N3, with a molecular weight of 319.27 and CAS number 4368-28-9, has the following molecular structure:

[0003] Compared to commonly used anesthetics, TTX has an anesthetic potency more than ten thousand times stronger and a longer duration of action, completely blocking withdrawal symptoms induced by naloxone after morphine withdrawal. Tetradixine exerts potent analgesic and anesthetic effects even in minute amounts in the body. However, TTX suffers from poor solubility, being only slightly soluble in weak acids, anhydrous ethanol, and ether, and almost insoluble in other organic reagents. Furthermore, it exhibits high activity, high toxicity, and a low therapeutic index, severely hindering its clinical research and application. The safety and stability of TTX remains a major obstacle. It is speculated that the unstable analgesic efficacy of monomeric TTX stems from the inability to determine an effective safe dosage. At excessively high doses, some TTX molecules cross the blood-brain barrier, producing some analgesic effect, but due to its high toxicity, it can cause severe hypotension and respiratory depression, thus compromising safety. Conversely, at excessively low doses, TTX molecules are completely blocked by the blood-brain barrier, failing to produce analgesia.

[0004] To overcome the aforementioned drawbacks of TTX, those skilled in the art have continuously developed TTX derivatives. However, it is clear that each technical route has its own disadvantages, such as high-burst drug release, the low mechanical strength of the gel leading to potential dose dumping, poor biocompatibility of polymer micelle systems, and the gradual decrease in pH value of the system due to acidic degradation, as well as the potential safety hazards to humans, the environment, and organisms posed by irritating small molecule acyl chloride raw materials. Although developing tetrodoxin conjugates from natural compound molecules has relatively high safety after hydrolysis, the number of selectable natural compound molecules is limited; while developing them from artificially synthesized molecules offers a wide range of selectable molecules, no relevant research results have been found on their application in the pharmaceutical technology of tetrodoxin. Further analysis of the characteristics of polymer carriers reveals that natural polymer groups (such as polymers derived from alginate) have good interactions with cell surface functional groups, stable molecular structures, are renewable and biodegradable, and have good biocompatibility (no obvious toxicity to organisms). However, they have limitations such as poor performance (mechanical properties, thermal stability, and chemical resistance are inferior to synthetic polymers), complex preparation processes, and high costs (extraction and modification require large amounts of energy and chemical reagents, and have high technical requirements). Although artificially synthesized polymer groups (such as carboxylated dextran, carboxylated β-cyclodextrin, and polyacrylic acid) may contain a certain proportion of isomers and the synthesis may use chemical photoinitiators, crosslinking agents, and surfactants that are toxic to organisms, the risks can be reduced through rational design and optimization. They also have a wider range of molecular selectivity and controllability, and can adjust the structure and properties as needed to meet specific drug delivery and release requirements. Therefore, they still have important significance and value in the pharmaceutical field.

[0005] Studies have shown that NGF, brain-derived nerve growth factor (BDNF), neurotrophin-3 (NT-3), IL-1β, TNF-α, interleukin (I-6), and transforming growth factor (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 contain a small amount of IL-1, and most cells can synthesize and secrete IL-1 only when subjected to certain exogenous stimuli. IL-1 exists in two forms: IL-1α and IL-1β. IL-1β is an important pro-inflammatory factor. Studies have confirmed (MH. van den Beuken-van Everdingen, L.M. Hochstenbach, E.A. Joosten, C.V. Tjan Heijnen, D.J. Janssen, Update on prevalence of pain in patients with cancer: systematic review and meta-analysis, Pain Symptom Manag. 51(2016)1070-1090.) that IL-1β expression is significantly increased in a rat model of bone cancer pain.

[0006] TNF-α is a pro-inflammatory cytokine mainly synthesized and secreted by macrophages and monocytes, and then produced through autocrine and paracrine processes. Experiments showing increased TNF-α production in many pathological conditions have revealed that TNF-α not only activates osteoclasts to participate in bone destruction but also plays an important role in pain hypersensitivity. Antagonizing TNF-α can reduce bone destruction and alleviate pain hypersensitivity. Studies on the correlation between TNF-α and cancer pain (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, pp. 299-307, 2007.) have found that TNF-α is also involved in the development and progression of cancer pain.

[0007] TTX has a wide range of potential applications, including neurological diseases, cancer pain, neuralgia, anesthesia, and heroin dependence. However, its current application is mainly focused on relieving pain in cancer patients. It is evident that the unresolved issues with existing TTX administration technologies limit its widespread use. Summary of the Invention

[0008] To address the shortcomings of current pharmaceutical technologies for tetrodoxin (TTX), one objective of this invention is to provide a tetrodoxin conjugate (PTTX), the general formula of which is: Wherein, R is the skeleton of a carboxyl-containing polymer compound, which is selected from carboxylated dextran, carboxylated β-cyclodextrin, alginate, or polyacrylic acid. Specific coupling compound subdivisions and structural characteristics are as follows:

[0009] Preferably, the structural formula of the tetrodocin coupling compound is selected from formulas (I), (II), (III), and (IV):

[0010] In formula (I), n = 1 to 20000;

[0011] In formula (II), R = COCH2CH2COOH or H.

[0012] In formula (Ⅲ), m = 1 to 2000, n = 1 to 1900;

[0013] In formula (Ⅳ), m = 1 to 9000 and n = 1 to 10000.

[0014] The tetrodocin conjugate of formula (I) is hereinafter referred to as PTTX1; the tetrodocin conjugate of formula (II) is hereinafter referred to as PTTX2; the tetrodocin conjugate of formula (III) is hereinafter referred to as PTTX3; and the tetrodocin conjugate of formula (IV) is hereinafter referred to as PTTX4.

[0015] A second objective of this invention is to provide a method for preparing the tetrodoxin conjugate. The method involves using a carboxyl-containing polymer and tetrodoxin as raw materials, adding a catalyst and solvent, and then obtaining the tetrodoxin conjugate through a coupling reaction. The carboxyl-containing polymer is selected from any one of carboxylated dextran, carboxylated β-cyclodextrin, alginate, or polyacrylic acid.

[0016] Preferably, the catalyst for the coupling reaction is selected from any one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N,N′-dicyclohexylcarboimide, in combination with N,N-dimethylaminopyridine or other organic bases.

[0017] Preferably, the solvent for the coupling reaction is selected from non-hydroxyl solvents, including but not limited to dimethyl sulfoxide or N,N-dimethylformamide.

[0018] A third objective of this invention is to provide the application of tetrodoxin conjugates, specifically for the preparation of sodium channel blocker pharmaceutical compositions.

[0019] Preferably, the application of the tetrodoxin conjugate includes using the tetrodoxin conjugate in the preparation of a pharmaceutical composition for relieving pain.

[0020] Preferably, the application of the tetrodoxin conjugate includes using the tetrodoxin conjugate in the preparation of a pharmaceutical composition for relieving neuropathic pain.

[0021] The fourth objective of this invention is to provide a tetrodoxin conjugate formulation, which is prepared by adding pharmaceutically acceptable excipients to the tetrodoxin conjugate.

[0022] Preferably, the tetrodoxin conjugate formulation is hydrolyzed and releases tetrodoxin upon entering the body; the drug window of the tetrodoxin conjugate formulation can be altered by selecting the molecular formula of the tetrodoxin conjugate and changing the type and / or quantity of the pharmaceutically acceptable excipients added.

[0023] 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 the following: water, physiological saline, acetic acid, gelatin, gum arabic, lactose, starch, cellulose, magnesium stearate, talc, vegetable oil, cyclodextrin, polyalkylene glycols, etc.

[0024] Preferably, the dosage form of the tetrodoxin conjugate is an injection.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] The present invention relates to tetrodoxin conjugate, which has analgesic effects associated with TTX. The use of high molecular weight conjugate increases the effective dose and the drug window, thus greatly improving the safety of medication. In addition, the natural high molecular weight has good biocompatibility and is biodegradable, and will not cause harm to human health. Attached Figure Description

[0027] Figure 1 shows the general formula of the PTTX of the present invention.

[0028] Figure 2.1 shows the 1H NMR spectrum of the dextran raw material in D2O.

[0029] Figure 2.2 shows the 1H NMR spectrum of the carboxylated dextran raw material in D2O.

[0030] Figure 2.3 shows the 1H NMR spectrum of alginate in D2O. 1 H NMR (400MHZ, D2O) δ: 3.5~5.2ppm.

[0031] Figure 2.4 shows the 1H NMR spectrum of TTX in 4% CD3COOD / 96% D2O. 1 H NMR (400MHZ, 4% CD3COOD / 96%D2O) δ: 5.45 (d, J = 9.4HZ, 1H), 4.26 (d, J = 1.8HZ, 1H), 4.22 (br, 1H), 4.05 (t, J=2.1HZ, 1H), 3.99~4.01 (m, 2H), 3.93 (s, 1H), 2.33 (d, J=9.5HZ, 1H).

[0032] Figure 2.5 shows the 1H NMR spectrum of PTTX1 in D2O, where the characteristic peaks of TTX can be observed.

[0033] Figure 2.6 shows the 1H NMR spectrum of PTTX3 in D2O.

[0034] Figure 2.7 shows the 1H NMR spectrum of PTTX4 in D2O.

[0035] Figure 3 shows the particle size distribution of PTTX1 in water (0.16 g / L). The horizontal axis SIZE represents the average particle size (nanometers), and the vertical axis represents the percentage of distribution.

[0036] Figure 4 shows the morphological changes of rats inoculated with MADB106 cells in the soles of their feet.

[0037] Figure 5.1 shows the cell viability of PC12 cells after incubation for 24 h in different concentrations of carboxylated dextran. The vertical axis represents cell viability (the ratio of cell viability at different polymer concentrations to that of the blank control cells) (100%), and the horizontal axis represents the concentration of carboxylated dextran (w / w).

[0038] Figure 5.2 shows the cell viability of C2C12 cells after incubation for 24 h in different concentrations of carboxylated dextran. The vertical axis represents cell viability (100%), and the horizontal axis represents the concentration of carboxylated dextran (w / w).

[0039] Figure 5.3 shows a diagram of live chromosomes and carboxylated dextran. Live cells are shown in green.

[0040] Figure 5.4 shows the cell viability of PC12 cells after incubation for 24 h in different concentrations of carboxylated β-cyclodextrin. The vertical axis represents cell viability (100%), and the horizontal axis represents the concentration of carboxylated β-cyclodextrin (w / w).

[0041] Figure 5.5 shows the cell viability of C2C12 cells after incubation for 24 h in different concentrations of carboxylated β-cyclodextrin. The vertical axis represents cell viability (100%), and the horizontal axis represents the concentration of carboxylated β-cyclodextrin (w / w).

[0042] Figure 5.6 shows a diagram of live chromosomes and carboxylated β-cyclodextrin. Live cells are shown in green.

[0043] Figure 5.7 shows the cell viability of PC12 cells after incubation in different concentrations of alginate for 24 h. The vertical axis represents cell viability (the ratio of cell viability at different polymer concentrations to that of the blank control cells) (100%), and the horizontal axis represents the concentration of alginate (w / w).

[0044] Figure 5.8 shows the cell viability of C2C12 cells after incubation in different concentrations of alginate for 24 h. The vertical axis represents cell viability (100%), and the horizontal axis represents the concentration of alginate (w / w).

[0045] Figure 5.9 shows the active chromosome-alginic acid diagram. Live cells are shown in green.

[0046] Figure 5.10 shows the cell viability of PC12 cells after incubation with different concentrations of polyacrylic acid for 24 h. The vertical axis represents the viability (100%), and the horizontal axis represents the concentration of polyacrylic acid (w / w).

[0047] Figure 5.11 shows the cell viability of C2C12 cells after incubation in different concentrations of polyacrylic acid for 24 h. The vertical axis represents the viability (100%), and the horizontal axis represents the concentration of polyacrylic acid (w / w).

[0048] Figure 5.12 shows a live chromosome-polyacrylic acid image. Live cells are shown as green.

[0049] Figure 6 shows the in vitro release curves of different TTX conjugates, where: Figure 6A shows the in vitro release curve of PTTX1 in PBS (pH=7.4); Figure 6B shows the in vitro release curve of PTTX2 in PBS (pH=7.4); Figure 6C shows the in vitro release curve of PTTX3 in PBS (pH=7.4); Figure 6D shows the in vitro release curve of PTTX4 in PBS (pH=7.4); Figure 6E shows the in vitro release curve of TDP-TTX in PBS (pH=7.4) [Source: Zhao C, Liu A, Santamaria CM, et al. Polymer-tetrodotoxin conjugates to induce prolonged duration local anesthesia with minimal toxicity[J]. Nature communications, 2019, 10(1):2566.].

[0050] Figure 7 shows the 1H NMR spectrum of tacrolimus, carboxylated dextran-tacrolimus, and carboxylated dextran in D2O, where the characteristic peaks of tacrolimus can be observed.

[0051] Figure 8 shows the 1H NMR spectra of anhydrous theophylline, carboxylated dextran-anhydrous theophylline, and carboxylated dextran in D2O, where the characteristic peaks of anhydrous theophylline can be observed.

[0052] Figure 9 shows the 1H NMR spectra of warfarin, carboxylated dextran-warfarin, and carboxylated dextran in D2O, where the characteristic peaks of warfarin can be observed.

[0053] Figure 10 shows the physiological state of mice after injection of PTTX1 and saline.

[0054] Figure 11 shows the physiological status of mice after injection of PTTX1 and saline.

[0055] Figure 12 shows the physiological status of mice after injection of PTTX1 and saline. Detailed Implementation

[0056] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0057] The tetrodoxin conjugate used in this invention was prepared by the applicant. All other experimental materials, unless otherwise specified, were obtained commercially. Alginic acid and polyacrylic acid were purchased from Merrill, polyvinyl alcohol from Aladdin, dextran, β-cyclodextrin, dimethyl sulfoxide, N,N-dimethylaminopyridine, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride from Anaiji, tetrodoxin from Huamu, and the TTX Elisa kit from Reagen. Experimental animals were obtained from Hefei Qingyuan Biotechnology Co., Ltd. The abbreviations used in this application are as follows:

[0058] Dimethyl sulfoxide: DMSO

[0059] N,N-Dimethylaminopyridine: DMAP,

[0060] 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride: EDC . HCl,

[0061] TTX (Tyzoxin)

[0062] Titridocin conjugate: PTTX (its general structural formula is shown in Figure 1).

[0063] In the following embodiments, a 0.6% acetic acid aqueous solution is prepared as follows: accurately measure 0.6 mL of acetic acid, add physiological saline for injection, and dilute to 100 mL. Shake well to obtain a 0.6% acetic acid aqueous solution (prepare immediately before use).

[0064] Example 1

[0065] Step 1: Prepare tertridoxin conjugate-1 (PTTX1). The synthetic route is as follows:

[0066] Step 1.1: Prepare carboxylated dextran.

[0067] The reaction raw materials and amounts included: 555.5 mg succinic anhydride, 205.6 mg dextran (molar mass: 20000 Da, its 1H NMR spectrum is shown in Figure 2.1), and 91.7 mg N,N-dimethylaminopyridine.

[0068] The above raw material was dissolved in anhydrous dimethyl sulfoxide under a nitrogen atmosphere and stirred at room temperature for more than 8 hours. After dialyzing, it was freeze-dried (-40℃, 72h) to obtain a white solid product, which was carboxylated dextran (its 1H NMR spectrum is shown in Figure 2.2).

[0069] Step 1.2, prepare PTTX1.

[0070] The reaction raw 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.

[0071] In a nitrogen atmosphere, TTX was dissolved and dispersed in anhydrous DMSO in a reaction flask, followed by the addition of other raw materials. The reaction was carried out at room temperature for 7 days to allow for complete reaction. After the reaction was completed, the reaction solution was dialyzed and lyophilized (-40℃, 72h) to obtain a white solid PTTX1. After washing and drying, the product weighed 71.54 mg, with a reaction yield greater than 81.5%.

[0072] The 1H NMR spectrum of PTTX1 is shown in Figure 2.5, and the characteristic peaks of TTX (its 1H NMR spectrum is shown in Figure 2.4) can be observed. An appropriate amount of PTTX1 was weighed and uniformly dispersed in water to prepare a 0.16 g / L solution, the particle size distribution of which is shown in Figure 3. Step 2: Preparation of the stock solution for tetrodoxin conjugate-1 (PTTX1) formulation.

[0073] Accurately weigh 40 mg of PTTX1 prepared in step one of this embodiment, then dissolve it in physiological saline to a final volume of 20 g / L to obtain the PTTX1 stock solution. Store at -20°C for later use.

[0074] Example 2

[0075] Step 1: Prepare tertridoxin conjugate-2 (PTTX2). The synthetic route is as follows:

[0076] Step 1.1 Preparation of carboxylated β-cyclodextrin.

[0077] The reaction raw materials and amounts include: 1.0 g β-cyclodextrin, 2.8 g succinic anhydride, and 0.45 g N,N-dimethylaminopyridine.

[0078] Under a nitrogen atmosphere, the above-mentioned raw materials were added to the reaction flask and dissolved in 10 mL of anhydrous DMSO. The mixture was stirred at room temperature for at least 8 hours. After the reaction was completed, the reaction solution was dialyzed using a dialysis bag (molar mass: 500 Da) for 24 hours. The dialysate was deionized water, with a volume of 3 L, and the water was changed 4 times. After dialysis, the solution was lyophilized (-40℃, 72 h) to obtain a white solid product, which was carboxylated β-cyclodextrin.

[0079] Step 1.2 Preparation of PTTX2.

[0080] The reactants and their quantities include: 53 mg (prepared in step 1.1) carboxylated β-cyclodextrin, 1.0 mg TTX, and 6.9 mg EDC. . HCl, 2.6 mg DMAP.

[0081] Under a nitrogen atmosphere, the above-mentioned raw materials were added to a reaction flask and dissolved in 5 mL of anhydrous DMSO. The reaction was carried out at room temperature for 7 days to allow for complete reaction. After the reaction was completed, the reaction solution was dialyzed using a dialysis bag (Mw: 1000 Da) with water for 24 h. The dialysate was deionized water, with a volume of 3 L, and the water was changed 4 times. After dialysis, the solution was lyophilized (-40 °C, 72 h) to obtain a white solid PTTX2. After washing and drying, the product weighed 45.00 mg, with a reaction yield greater than 83%.

[0082] Step 2: Preparation of stock solution for tetrodoxin conjugate-2 (PTTX2) formulation

[0083] Accurately weigh 5.0 mg of PTTX2 prepared in step one of this embodiment, then dissolve it in physiological saline to a final volume of 5 g / L to obtain the PTTX2 stock solution. Store at -20°C for later use.

[0084] Example 3

[0085] Step 1: Prepare tertridoxin conjugate-3 (PTTX3). The synthetic route is as follows:

[0086] In a nitrogen atmosphere, TTX (0.6 g) and EDC were dissolved and dispersed in DMSO (1.5 L) in a reaction flask. . HCl (0.76 g), DMAP (46 mg), and alginate (8.9 g, its 1H NMR spectrum is shown in Figure 2.3) were reacted at room temperature for 5 to 7 days to allow for complete reaction. After the reaction, the reaction solution was dialyzed against water for 24 h using a dialysis bag (Mw: 1000 Da). After dialysis, the solution was lyophilized (-40 °C, 72 h). The product obtained by lyophilization was a white solid PTTX3. After washing and drying, the weight was 8.10 g, and the reaction yield was greater than 85%. Figure 2.6 shows the 1H NMR spectrum of PTTX3 in D2O. The peak positions of TTX and alginate overlapped when the chemical shift was below 5.2 ppm. The characteristic peak of TTX (its 1H NMR spectrum is shown in Figure 2.4) could be observed at 5.45 ppm.

[0087] Step 2: Preparation of stock solution for tetrodoxin conjugate-3 (PTTX3)

[0088] Accurately weigh 5 mg of PTTX3 prepared in step one of this embodiment, then dissolve it in physiological saline to a final volume of 5000 mg / L (5 g / L) to obtain the PTTX3 stock solution. Store at -20°C for later use.

[0089] Example 4

[0090] Step 1: Prepare tertridoxin conjugate-4 (PTTX4). The synthetic route is as follows:

[0091] In a nitrogen atmosphere, TTX (0.6 g), EDCHCl (0.76 g), DMAP (46 mg), and polyacrylic acid (1 g) were dissolved and dispersed in DMSO (1.5 L) in a reaction flask and reacted at room temperature for 5 to 7 days to allow for complete reaction. After the reaction was complete, the reaction solution was dialyzed against water for 24 h using a dialysis bag (Mw: 1000 Da). After dialysis, the solution was lyophilized (-40 °C, 72 h). The product obtained by lyophilization was a white solid PTTX4. After washing and drying, the product weighed 1.50 g, and the reaction yield was greater than 90%. Figure 2.7 shows the 1H NMR spectrum of PTTX4 in D2O, where the characteristic peak of TTX can be observed at 5.45 ppm.

[0092] Step 2: Preparation of stock solution for tetrodoxin conjugate-2 (PTTX4) formulation

[0093] Accurately weigh 5 mg of PTTX4 prepared in step one of this embodiment, then dissolve it in physiological saline to a final volume of 5000 mg / L (5 g / L) to obtain the PTTX4 stock solution. Store at -20°C for later use.

[0094] Comparative Example 1

[0095] Synthesis of carboxylated dextran-coupled drugs.

[0096] The reaction materials and quantities included: 155 mg (prepared in step 1.1) of carboxylated dextran, 14.4 mg of EDCHCl, 0.92 mg of DMAP, 12 mg of tacrolimus, 12 mg of anhydrous theophylline, and 12 mg of warfarin. Tacrolimus, anhydrous theophylline, and warfarin were all purchased from Aladdin.

[0097] Tacrolimus was dissolved and dispersed in anhydrous DMSO in a reaction flask under a nitrogen atmosphere. Other starting materials were then added, and the reaction was carried out at room temperature for one week. After the reaction was complete, the reaction solution was dialyzed and lyophilized (-40°C, 72 h) to obtain a white solid, carboxylated dextran-tacrolimus, which weighed 100 mg after washing and drying. The synthesis of carboxylated dextran-anhydrous theophylline followed the same steps, yielding a final product of 140 mg. The synthesis of carboxylated dextran-warfarin followed the same steps, yielding a final product of 130 mg.

[0098] The above experiments show that carboxylated dextran can stably couple different drugs as a carrier, that is, the drugs can be slowly released through ester bond hydrolysis, which verifies the stability of the carrier.

[0099] Comparative Example 2

[0100] Synthesis of TDP-TXX drug conjugate.

[0101] 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 the mixture was allowed to stand at room temperature for 24 h. A solution of anhydrous DMSO (10 mL) containing TTX (1 mg, 0.003 mmol) was added, and the mixture was allowed to stand at room temperature for 7 days to allow for complete reaction. After the reaction was complete, 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 lyophilized overnight. Subsequently, the dried polymer was redissolved in DCM and purified by precipitation with 30 mL of diethyl ether. The supernatant was then discarded, and the precipitate was vacuum dried for at least 8 hours. The resulting pale yellow solid product was the TDP-TXX drug conjugate. The dried TDP-TXX drug conjugate was stored in a desiccator for further use. The TDP mentioned above is a poly(triol dicarboxylic acid)-co-poly(ethylene glycol) polymer.

[0102] The samples prepared in the above examples and comparative examples were subjected to the following experimental cases.

[0103] Experimental Example 1

[0104] Step 1.1: Take the PTTX1 preparation stock solution with a concentration of 20 g / L (mg / mL) obtained in Example 1 out of the refrigerator, and when it returns to room temperature, add physiological saline for injection to prepare PTTX1 preparations of different concentrations (0.625 g / L to 16 g / L), and store them in the refrigerator for later use.

[0105] Step 1.2: Take the PTTX2 formulation mother liquor with a concentration of 5 g / L obtained in Example 2 out of the refrigerator, and after it returns to room temperature, obtain PTTX2 formulations of different concentrations (0.625 g / L to 16 g / L) using the same method as above.

[0106] Step 1.3: Take the PTTX3 preparation stock solution with a concentration of 5 g / L obtained in Example 3 out of the refrigerator, and when it returns to room temperature, add physiological saline for injection to prepare PTTX3 preparations of different concentrations (1250 mg / L to 39.0625 mg / L), and store them in the refrigerator for later use.

[0107] Step 1.4: Add PTTX4 stock solution with a concentration of 5000 mg / L to injectable physiological saline to prepare PTTX4 preparation with a concentration of 1250 mg / L to 39.0625 mg / L, and conduct toxicity tests on PTTX4 preparation.

[0108] Step 1.5: Accurately weigh 1 mg of TTX, dissolve it in 100-200 μL of 0.6% acetic acid aqueous solution, add physiological saline for injection, and make up to 10 mL to obtain a TTX preparation stock solution with a concentration of 100 mg / L. Store at -20℃ for later use.

[0109] Take out the 100 mg / L TTX stock solution from the refrigerator and let it return to room temperature. Add physiological saline for injection to prepare TTX preparations of different concentrations (6 mg / L to 0.125 mg / L) and store them in the refrigerator for later use.

[0110] Step 1.6: Mice were used as experimental animals, weighing 20g ± 2g, with an equal number of males and females. Mice were acclimatized for 4–7 days before the experiment. They were fasted for 12 hours before the experiment, but had free access to water. After fasting for 12 hours, the mice were randomly divided into groups of 10.

[0111] Step 1.7: After fasting for 12 hours, take one group (10 mice) and inject approximately 40 μL of the PTTX formulation prepared in steps 1.1 to 1.4 above into the intramuscular muscle of the thigh of each mouse, at a dose of 2 μL / g body weight. Leakage must be avoided during injection. Observe the mice for 20-30 minutes after injection to assess their survival. If more than half of the mice die, reduce the concentration and repeat the experiment. If less than half die, increase the concentration and repeat the experiment until a PTTX formulation concentration with a 50% lethality (LD50) is determined.

[0112] Step 1.8: After fasting for 12 hours, remove one group (10 mice) and inject approximately 40 μL of the 6 mg / L TTX preparation prepared in the previous step into the intramuscular muscle of the thigh, at a dose of 2 μL / g body weight. Leakage must be avoided during injection. Observe for 20-30 minutes to assess mouse survival. If more than half of the mice die, the concentration should be reduced and the experiment repeated. If less than half die, the concentration should be increased and the experiment repeated until a TTX concentration with a 50% lethality (LD50) is determined.

[0113] Step 1.9: Referring to Step 1.8, after fasting for 12 hours, take one group (10 mice) and inject approximately 40 μL of TDP-TTX formulation into the intramuscular muscle of the mouse's thigh at a dose of 2 μL / g body weight. Leakage must be avoided during the injection. Observe for 20-30 minutes to assess mouse survival. If more than half of the mice die, the concentration should be reduced and the experiment repeated. If less than half die, the concentration should be increased and the experiment repeated until a TDP-TTX formulation concentration with a 50% lethality (LD50) is determined. The experimental results are shown in Table 1 below.

[0114] Table 1: Toxicity Test Results (LD50)

[0115] The above experiments show that the LD50 concentrations of PTTX1, PTTX2, and TDP-TTX formulations are all significantly higher than the 4.0 mg / L of TTX formulation. The LD50 concentration of PTTX3 formulation is 206 mg / L, the LD50 concentration of PTTX4 formulation is 500 mg / L, and the LD50 concentration of TDP-TTX formulation is 19400 mg / L, indicating that the safety of PTTX1, PTTX2, PTTX3, PTTX4, and TDP-TTX formulations is significantly higher than that of TTX formulation.

[0116] Experiment Example 2

[0117] Step 2.1: The preparation of different concentrations of drugs (PTTX1, PTTX2, PTTX3, PTTX4, TTX, and TDP-TTX) was the same as in Experiment 1. Physiological saline injection was used as the blank control group.

[0118] Step 2.2: Mice were used as experimental animals, weighing 20g ± 2g, with an equal number of males and females. Mice were acclimatized for 4–7 days before the experiment. They were fasted for 12 hours before the experiment, but had free access to water. After fasting for 12 hours, the mice were randomly divided into groups of 10.

[0119] Step 2.3: After fasting for 12 hours, remove one group (n=10) of mice and inject them with different concentrations of the drug (approximately 40 μL) intramuscularly at a dose of 2 μL / g body weight. The blank control group was injected with physiological saline at a dose of 2 μL / g body weight. Leakage must be avoided during the injection process. Timing begins 40 minutes after the drug or saline injection is completed. Then, inject a 0.6% acetic acid solution into the peritoneum of the mice.

[0120] Step 2.4: After the acetic acid aqueous solution injection, place the mice separately and observe and record the number of writhing movements in each mouse within 15 minutes. A positive writhing response is defined as abdominal retraction, trunk and hind limb extension, and raised buttocks. Calculate the pain inhibition rate of the corresponding concentration of the preparation for each group based on the average number of writhing movements. The calculation formula is as follows:

[0121] Pain suppression rate (%) = (average number of writhing movements in the blank control group - average number of writhing movements in the drug-treated group) / average number of writhing movements in the blank control group × 100%.

[0122] Different formulation concentrations that achieved a 50% pain inhibition rate (EC50) were determined by increasing or decreasing the concentration. The results are shown in Tables 2 and 3.

[0123] Table 2: Results of the analgesia trial (EC50)

[0124] Table 3. Mouse Acetic Acid Writhing Test

[0125] The data in Tables 2 and 3 show that PTTX1, PTTX2, PTTX3, and TDP-TTX have analgesic efficacy comparable to TTX. Compared with Table 1, the pharmacoscopic windows of PTTX1, PTTX2, PTTX3, PTTX4, and TDP-TTX are significantly longer than those of TTX. The data indicate that the concentration of TDP-TTX used is 3.12 times that of PTTX1, its LD50 is 94 times that of PTTX3, and its EC50 is 40 times that of PTTX3. This suggests that the active ingredient TTX loaded in TDP-TTX is too low (0.1–1.6 μg / mg in the literature). Furthermore, TDP is synthesized from polyethylene glycol (PEG), sebacic acid, and glycerol. Previous literature reports that treatment with PEGylated drugs in patients who have acquired anti-PEG antibodies can lead to accelerated blood clearance, reduced drug efficacy, hypersensitivity reactions, and in some cases, life-threatening side effects.

[0126] Experimental Example 3

[0127] PTTX in vitro release assay procedure: Accurately weigh 1.0 mg of PTTX1, PTTX2, PTTX3, and PTTX4 prepared in the example, dissolve them in 100 μL of PBS (pH=7.4) to prepare a solution with a concentration of 10 g / L, and add 100 μL of each solution to a dialysis bag (molar mass: 1000 Da) and seal the bag. Place the dialysis bag in a sample vial containing 10 mL of PBS (pH=7.4) and tighten the cap. Then place the sample vial in a constant temperature shaking incubator (37℃, 90 rpm). Samples were taken at 0.5 h, 1 h, 3 h, 6 h, 9 h, 12 h, 24 h, 2 d, 3 d, 5 d, and 7 d. After sampling, the release amount was measured using a TTX ELISA kit, and the results are shown in Figure 6.

[0128] Figure 6 shows the in vitro release curves of PTTX. Comparing these results with the TDP-TTX reported in the reference, in Figure 6A, PTTX1 releases 429.0 ng of TTX at 72 h and 543.65 ng at 168 h. In Figure 6B, PTTX2 releases 240.9 ng of TTX at 72 h and 248.7 ng at 168 h. In Figure 6C, PTTX3 releases 446.5 ng of TTX at 72 h and 482.2 ng at 168 h. In Figure 6D, PTTX4 releases 313.9 ng of TTX at 72 h and 408.7 ng at 168 h. Figure 6E allows for the estimation of the TDP-TTX released (based on literature reports).g D8P 200 The TTX conjugate (TDP-TTX with a TTX loading of 0.31 μg / mg) releases approximately 46.5 ng of TTX at 72 h and approximately 86.8 ng of TTX at 168 h. Due to the low release of TDP-TTX, its effective anesthesia when injected into the sciatic nerve of mice has been observed in the literature. However, systemic administration may not reach the effective dose, thus failing to produce an anesthetic effect.

[0129] Experiment Example 4

[0130] 4.1 Cell Culture

[0131] Cell culture of C2C12 mouse myoblasts (Wuhan Pronosai) and PC12 rat adrenal pheochromocytoma cells (ATCC, CRL-1722) (Wuhan Pronosai).

[0132] C2C12 cells were cultured in DMEM (bkmam) containing 20% ​​FBS (Gibco) and 1% penicillin-streptomycin (Shanghai Yuanye Biotechnology Co., Ltd.). Cells were seeded at 50,000 cells / mL into 24-well plates and incubated in DMEM containing 2% horse serum (Wuhan Pronosai) and 1% penicillin-streptomycin for 10–14 days to differentiate into myotubules.

[0133] PC12 cells were grown in DMEM containing 12.5% ​​horse serum, 2.5% FBS, and 1% penicillin-streptomycin. Cells were seeded into 24-well plates, and 50 ng / mL nerve growth factor (Wuhan Pronosai) was added 24 hours after seeding.

[0134] 4.2 Cell viability

[0135] Cells (1x10) 4 Cells were incubated for 24 hours with various concentrations of carboxylated dextran, carboxylated β-cyclodextrin, alginate, and polyacrylic acid. After incubation, cells were washed five times with PBS to remove polymers, and cell viability was determined by MTT assay. In summary, culture supernatants from control wells and polymer-containing wells were collected, and cells were incubated with MTT (0.5 mg / mL, 3 h). The resulting polymer was dissolved in 200 μL DMSO, and the absorbance was measured at 550 nm. The absorbance of control wells was assumed to be 100%, and cell viability in the treated wells relative to the control wells was determined.

[0136] 4.3 Results of cell viability assay

[0137] Figures 5.1 and 5.2 show cell viability at different concentrations of carboxylated dextran and the viability of the blank control. Figures 5.4 and 5.5 show cell viability at different concentrations of carboxylated β-cyclodextrin and the viability of the blank control. Figures 5.7 and 5.8 show cell viability at different concentrations of alginate and the viability of the blank control. Figures 5.10 and 5.11 show cell viability at different concentrations of polyacrylic acid and the viability of the blank control. No significant differences were observed in any of these values, indicating that the polymers carboxylated dextran, carboxylated β-cyclodextrin, alginate, and polyacrylic acid do not reduce cell viability and exhibit good cell compatibility.

[0138] Figures 5.3, 5.6, 5.9, and 5.12 show that all living cells are green, further demonstrating that carboxylated dextran, carboxylated β-cyclodextrin, alginate, and polyacrylic acid are almost non-toxic.

[0139] Experimental Example 5

[0140] 5.1 Establishment of a rat model of skin cancer pain:

[0141] (1) Laboratory animals

[0142] Healthy adult female (SD) rats, weighing 140-150g, were purchased from Jiangsu Wukong Pharmaceutical Co., Ltd. They were housed individually in cages, with ample food and water provided. The laboratory maintained constant temperature and humidity, and rats had free access to food and water. This experiment strictly adhered to the requirements of the International Association for the Study of Pain's guidelines for pain research using conscious animals. Furthermore, laboratory operating procedures were strictly followed throughout the experiment.

[0143] (2) Experimental cells

[0144] The MADB106 rat breast cancer cell line was purchased from Shanghai Yuhao Biotechnology Co., Ltd.

[0145] (3) Preparation and grouping of rat skin cancer pain model

[0146] 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 five rats in each group. The rats in the model group were first anesthetized by intraperitoneal injection of 10% chloral hydrate. The skin of the rat paw pads was disinfected with povidone-iodine. 100 μL of MADB106 breast cancer cell suspension was slowly injected subcutaneously into the paw pads of the rats in the model group using a 1 mL syringe. Povidone-iodine was then applied locally for 1 minute. After the experiment, the rats were allowed to regain consciousness and were returned to their original cages for continued rearing. The rats were then closely observed for any adverse reactions.

[0147] As shown in Figure 4, the rats in the model group developed varying degrees of redness and swelling on the soles of their feet starting 3 days after inoculation, and the redness and swelling became more pronounced as the experiment progressed. This is because the tumor cells were present and continued to grow. If the redness and swelling were caused by inflammation, the rats would also initially show redness and swelling on the soles of their feet, but this would subside by day 10.

[0148] 5.2 Mechanical pain sensitivity testing:

[0149] Comparison of the mechanical paw withdrawal threshold (MPWT) of the operated hind paw 20 days after rat modeling showed that the MPWT was lower in the model group. There was a statistically significant difference between the model group and the control group (P<0.05).

[0150] Table 4 Comparison of MPWT values ​​of the hind paw on the operated side of rats 20 days after modeling. Note: *P<0.05 compared with the control group.

[0151] 5.3 Expression levels of inflammatory factors:

[0152] In the cytokine assay, skin tissue from both the control and model groups of the experimental animals was collected at day 20 for cytokine concentration determination. The concentrations of IL-1β and TNF-α were detected using an IL-1β ELISA kit (Lianke Biotechnology) and a TNF-α ELISA kit (Lianke Biotechnology), respectively. The experimental procedures were performed according to the manufacturer's instructions.

[0153] The experimental results are as follows: after rats were inoculated with tumor cells, the levels of IL-1β and TNF-α in the tumor-bearing skin tissue were higher than those in the blank control group (both P<0.05).

[0154] Table 5 Comparison of IL-1β and TNF-α expression levels in the skin tissues of two groups of rats (pg / mL) Note: *P<0.05 compared with the control group.

[0155] In this experiment, using an enzyme-linked immunosorbent assay (ELISA) to detect the concentrations of cytokines (IL-1β and TNF-α) in rat skin tissue in a tumor cell-bearing skin cancer pain model, we found that the concentrations of IL-1β and TNF-α in the model group were significantly higher than those in the control group on day 20 after tumor cell inoculation. Statistical comparison showed that the cytokine concentrations after tumor cell inoculation were significantly different from those in the control group (P < 0.05). This study demonstrates increased expression of IL-1β and TNF-α in tumor-bearing skin tissue, suggesting that inflammatory factors may be involved in tumor cell-induced hyperalgesia. Because the rat skin cancer pain model effectively simulates the pathological processes related to cancer pain, this model will contribute to elucidating the mechanisms of cancer pain and developing related treatments.

[0156] 5.4 Post-drug administration mechanical pain sensitivity test:

[0157] The study verified that intraperitoneal injection of the tetrodoxin conjugate provided by this invention can alleviate mechanical hyperalgesia in a cancer pain model and prolong drug release time. Twenty days after modeling, rats were intraperitoneally injected with the tetrodoxin conjugates prepared in Examples 1 to 4 of this invention. The MPWT of the rat paws was measured using an electronic Von Frey method at 1 hour before administration and at 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, and 12 hours after administration. Administration was based on the EC50 of PTTX1, PTTX2, PTTX3, PTTX4, TDP-TTX, and TTX. Rats were placed in an acrylic box with a perforated metal mesh bottom. After acclimatization for 20 minutes, measurements were started while the rats were at rest. The Von Frey wire stimulated the rat paw skin from bottom to top. Stimulation automatically stopped when a rapid paw withdrawal response occurred. Pressure values ​​were recorded, and a total of 5 measurements were taken at 3-minute intervals, with the average value taken. After the experiment, the rats were returned to their cages for continued rearing. The rats were in good condition, and no deaths occurred after 30 days of rearing.

[0158] As shown in Table 6, intraperitoneal injection of TTX significantly increased the MPWT of model rats, with analgesia starting 0.5 h after administration and reaching peak efficacy, which disappeared after 4 h. Injection of TDP-TTX had some analgesic effect, but the efficacy was not significant. Intraperitoneal injection of PTTX1 (carboxylated dextran-TTX) showed analgesia starting 0.5 h after administration, reaching peak efficacy at 2 h, with an analgesic duration of up to 12 h, and still exhibiting analgesic effects. It is reasonable to speculate that PTTX1 continued to provide analgesia. Intraperitoneal injection of PTTX2 (carboxylated β-cyclodextrin-TTX) showed analgesia starting 0.5 h after administration, reaching peak efficacy at 4 h, with analgesia lasting up to 12 h. Injection of PTTX3 (alginic acid-TTX) showed analgesia starting 0.5 h after administration, reaching peak efficacy at 1 h, followed by a slight decrease in efficacy, reaching a second peak efficacy at 6 h, with analgesic effects lasting for more than 12 h. Intraperitoneal injection of PTTX4 (polyacrylic acid-TTX) provides analgesia starting 0.5 hours after administration, maintaining a relatively mild analgesic effect until the effect disappears after 12 hours. PTTX1, PTTX2, PTTX3, and PTTX4 all have a longer duration of analgesia than TTX, and their analgesic efficacy is significantly higher than that of TDP-TTX.

[0159] Table 6. Changes in the mechanical withdrawal threshold of the cancer pain model after intraperitoneal injection of the polymer conjugate.

[0160] In summary, the polymer-TTX conjugates PTTX1 (carboxylated dextran-TTX), PTTX2 (carboxylated β-cyclodextrin-TTX), PTTX3 (alginic acid-TTX), and PTTX4 (polyacrylic acid-TTX) provided in this application can significantly improve the systemic analgesia treatment index, and the hydrolysis products (carboxylated dextran or carboxylated β-cyclodextrin) are not cytotoxic.

[0161] PTTX conjugates can significantly extend the therapeutic window of TTX, and in cancer pain models, all PTTX conjugates can significantly prolong the analgesic release time, with analgesia time more than 3 times that of TTX, without systemic toxicity.

[0162] The disadvantage of TTX lies in its very narrow therapeutic window, necessitating control of its release rate to avoid systemic toxicity while prolonging efficacy. The PTTX conjugates provided in this application precisely control the hydrolysis rate of the ester bond to maintain drug levels within the therapeutic window and release TTX above the effective dose. For example, PTTX1 releases 543.65 ng of TTX in 168 h, PTTX3 releases 482.2 ng of TTX in 168 h, and PTTX4 releases 408.7 ng of TTX in 168 h. In contrast, TDP-TTX in the literature only releases approximately 86.8 ng of TTX in 168 h. Furthermore, the low release of TDP-TTX necessitates local administration to achieve the desired efficacy; systemic administration may be ineffective.

[0163] Animal studies 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, 25 times that of PTTX2, 40 times that of PTTX3, and 47.67 times that of PTTX4. This means that the effective dose of TDP-TTX needs to be at least 3 times that of PTTX1, 25 times that of PTTX2, at least 40 times that of PTTX3, and at least 47.67 times that of PTTX4. Excessive dosage is problematic. Furthermore, PTTX is highly soluble in water and PBS, while TDP-TTX itself has extremely poor solubility, making it difficult to achieve an effective dose. It requires PEG to form an injectable formulation. Previous literature reports that treatment of patients with PEG-modified drugs who have acquired anti-PEG antibodies can lead to accelerated blood clearance, reduced drug efficacy, hypersensitivity reactions, and in some cases, life-threatening side effects.

[0164] Furthermore, in a cancer pain model experiment, this application conducted an analgesic experiment in rats via systemic administration (intraperitoneal injection) to verify the analgesic effect of PTTX and assess its safety. The experimental results showed that PTTX significantly reduced pain in rats with skin cancer induced by MADB106 breast cancer cells. The analgesic duration was as long as 12 hours or even longer. No animal deaths or signs of acute toxicity were observed during the experiment. In the literature, TDP-TTX is injected locally into the sciatic nerve of experimental mice to exert its therapeutic effect. In this experiment, TDP-TTX was also administered via intraperitoneal injection, but the analgesic effect was not significant and was far lower than that of PTTX. This demonstrates that systemic administration of TDP-TTX fails to achieve analgesic efficacy and its safety warrants further consideration.

[0165] Experimental Example 6

[0166] Step 1: Pain model establishment.

[0167] S1: Establishment of a preserved nerve injury (SNI) model.

[0168] S1.1: Before the operation, all surgical instruments are disinfected by soaking in 75% alcohol.

[0169] S1.2: Mice were deeply anesthetized with isoflurane, and their body temperature was maintained by covering them with sterile gauze during anesthesia.

[0170] S1.3: Use surgical scissors to remove hair from the left hind leg thigh area of ​​the mouse, and disinfect the exposed skin with iodine and 75% alcohol in sequence.

[0171] S1.4: Make an incision of about 1 cm on the lateral side of the left hind limb knee joint, and gently separate the muscle layer tissue with sterile forceps moistened with physiological saline.

[0172] S1.5: Use a glass needle to expose the sciatic nerve and its three terminal branches (common peroneal nerve, tibial nerve, and sural nerve).

[0173] S1.6: The peroneal nerve and tibial nerve were ligated with non-absorbable 4-0 chromium catgut. Slight twitching of the left hind limb was observed after ligation, indicating that the ligation was effective.

[0174] S1.7: Cut the two nerves about 2mm distal to the ligation site, taking care to avoid damaging the sural nerve.

[0175] S1.8: Gently reposition the severed nerve stump and suture the muscle layer and skin incision in sequence.

[0176] S2: Establishment of a bone cancer pain model.

[0177] S2.1: Mice were used as experimental animals, weighing 20g±2g. The mice were acclimatized for 4-7 days before the experiment began.

[0178] S2.2: Before the operation, all surgical instruments are sterilized by high temperature and high pressure and disinfected with 75% alcohol.

[0179] S2.3: Mice were deeply anesthetized with isoflurane, and their body temperature was maintained by covering them with sterile gauze during anesthesia.

[0180] S2.4: Prepare the skin around the knee joint on the left hind limb of the mouse, and disinfect the exposed skin with iodine and 75% alcohol in sequence.

[0181] S2.5: Make a longitudinal skin incision of about 0.5 cm above the right tibial plateau, adjacent to the patellar ligament, bluntly separate the fascia and muscles, and fully expose the surface of the upper tibial shaft.

[0182] S2.6: Use a 1mL syringe needle to drill a micro-hole with a diameter of about 0.1mm in the middle of the tibial shaft, taking care to avoid damaging the contralateral cortex.

[0183] S2.7: Using a microsyringe, aspirate 5 μL of lung cancer (LLC) cell suspension (containing approximately 2 × 10^4 cells) and slowly inject it into the tibial bone marrow cavity through the burr hole. The injection should last for about 1 minute to prevent the cell suspension from spilling out.

[0184] S2.8: After injection, seal the bone hole with sterile bone wax and suture the fascia and skin incision in sequence.

[0185] S3: Establishment of an arthritis pain model.

[0186] S3.1: Mice were used as experimental animals, weighing 20g±2g, with half males and half females. The mice were acclimatized for 4-7 days before the experiment began.

[0187] S3.2: Remove the Complete Freund's Adjuvant (CFA) from the refrigerator and allow it to return to room temperature before shaking it thoroughly.

[0188] S3.3: Mice were briefly anesthetized by inhalation with isoflurane, and the anesthesia was stopped after they lost consciousness.

[0189] S3.4: Prepare the skin around the ankle joint on the right hind limb of the mouse, and disinfect the exposed skin with 75% alcohol.

[0190] S3.5: Using a 0.5 mL insulin syringe, slowly inject 20 μL of CFA into the joint cavity of the left hind limb ankle.

[0191] S3.6: After injection, gently press the injection site for a moment to prevent CFA leakage.

[0192] Step 2: Single-dose mechanical pain threshold test (von Frey filament test).

[0193] S6.1: Two days before the formal test, place the mice alone in a transparent plastic observation box with a wire mesh bottom to allow them to adapt to the environment for at least 60 minutes, and then return them to the rearing cage.

[0194] S6.2: On the day of the formal test, place the mice back in the same testing environment and allow them to adapt for more than 30 minutes until they become quiet and show no obvious grooming or exploratory behavior.

[0195] S6.3: The injection methods and dosages for different pain models are as follows.

[0196] In the SNI (sparing nerve injury) model group, mice were injected intraperitoneally (subcutaneously) with the following doses of PTTX1: 0.25 mg / kg, 1 mg / kg, 2 mg / kg, and 2.5 mg / kg; PTTX2: 0.05 mg / kg, 0.1 mg / kg, 0.25 mg / kg, and 0.28 mg / kg; PTTX3: 0.05 mg / kg, 0.15 mg / kg, and 0.2 mg / kg; and PTTX4: 0.025 mg / kg, 0.05 mg / kg, 0.1 mg / kg, and 0.15 mg / kg.

[0197] In the bone cancer pain model group, mice were injected intraperitoneally (subcutaneously) with the following doses: PTTX1 2.5 mg / kg; PTTX2 0.28 mg / kg; PTTX3 0.20 mg / kg; and PTTX4 0.15 mg / kg.

[0198] In the arthritis pain model group, mice were injected intraperitoneally (subcutaneously) with the following doses: PTTX1 2.5 mg / kg; PTTX2 0.28 mg / kg; PTTX3 0.20 mg / kg; and PTTX4 0.15 mg / kg.

[0199] S6.4: The testing time points were selected as 1 hour before drug administration and 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, and 5 hours after drug administration. Mice were stimulated in ascending order during the test.

[0200] S6.5: For each test, use a series of standard von Frey filaments (stimulation force gradient range: 0.02-1g). Start with the lowest force filament and apply it vertically to the outer surface of the left hind paw of the mouse, allowing the filament to bend slowly and continue stimulating for 2-3 seconds. Apply stimulation 5 times consecutively to the same area of ​​the same paw, with each stimulation spaced approximately 10-15 seconds apart.

[0201] S6.6: Observe and record whether the mouse exhibits positive responses such as rapid paw retraction, paw licking, or violent paw shaking. If at least 3 out of 5 stimuli produce positive responses, the filament strength is determined to be the positive threshold.

[0202] If the positive threshold is not reached, replace the filament with the next increasing strength and repeat S6.5 until the positive threshold is measured.

[0203] S6.7: The interval between tests for each group of mice should be no less than 5 minutes to avoid behavioral adaptation or sensitization in the animals.

[0204] S6.8: Record the minimum filament force (grams, g) required to elicit a positive response in each mouse, and use this value as the mechanical pain threshold for that mouse. A higher value indicates lower pain sensitivity and better analgesic effect.

[0205] S6.9: The mechanical pain threshold of each group of mice was averaged. The SNI (sparing nerve injury) model group was recorded in Tables 7 to 10. The bone cancer pain model group was recorded in Table 11. The arthritis pain model group was recorded in Table 12.

[0206] Table 7 Analgesia after a single dose of PTTX1 in the SNI model group

[0207] Table 8. Analgesia after a single PTTX2 dose in the SNI model group

[0208] Table 9. Analgesia after a single dose of PTTX3 in the SNI model group.

[0209] Table 10 Analgesia after a single dose of PTTX4 in the SNI model group

[0210] Table 11 Analgesia after a single PTTX dose in the bone cancer pain group

[0211] Table 12 Analgesia with a single PTTX dose in the arthritis group

[0212] The above experiments showed that PTTX1, PTTX2, PTTX3, and PTTX4 had dose-dependent analgesic effects on SNI-induced neuropathic pain in mice: PTTX1 at high doses (2.5 mg / kg) had the strongest analgesic effect and the longest duration of action (4 hours); medium doses (2.0 mg / kg, 1 mg / kg) had transient analgesic effects, but the duration of action decreased with decreasing dose; low doses (0.25 mg / kg) had no significant analgesic effect. PTTX2 had the strongest analgesic effect at a dose of 0.28 mg / kg and the longest duration of action (3 hours); PTTX3 had the strongest analgesic effect at a dose of 0.20 mg / kg; and PTTX4 had the strongest analgesic effect at a dose of 0.15 mg / kg, with the longest duration of action (4 hours). They also exhibited similar analgesic effects in bone cancer and arthritis models.

[0213] It is evident that the PTTX conjugate provided by this invention can effectively alleviate chronic pain in mice at appropriate doses, and has potential clinical application value.

[0214] Experimental Example 7

[0215] A preserved nerve injury (SNI) model was established in mice using the same method as step S1 in Experiment Example 6.

[0216] In mice, PTTX1 was injected intraperitoneally (subcutaneously) at a dose of 2.5 mg / kg; PTTX2 at a dose of 0.28 mg / kg; PTTX3 at a dose of 0.20 mg / kg; PTTX4 at a dose of 0.15 mg / kg; and TTX at a dose of 8 μg / kg. The injections were administered twice daily for four consecutive days.

[0217] Before administration and at fixed times each day after administration, the mechanical pain threshold of each group of mice was tested and recorded according to steps S6.5 to S6.8 of Experiment Example 6, as shown in Table 13.

[0218] Table 13 SNI Model: Continuous Drug Administration Analgesia

[0219] Experiments showed that in SNI-induced neuropathic pain in mice, long-term administration of high-dose PTTX1 (2.5 mg / kg) significantly increased the mechanical pain threshold from day 1 after administration and maintained a high level for 1-9 days, demonstrating strong and long-lasting analgesic effects. PTTX2, PTTX3, and PTTX4 all showed a significant increase in the mechanical pain threshold from day 1 after administration. PTTX2 provided long-lasting analgesia for 9 days, while PTTX3 and PTTX4 lasted for up to 11 days. Although TTX provided analgesia for up to 13 days, its analgesic efficacy was only half that of PTTX1. Therefore, the PTTX conjugates provided by this invention can effectively alleviate neuropathic pain with long-term application and have potential long-term analgesic value.

[0220] Experimental Example 8

[0221] Pharmacokinetic assay (ELISA). Mice were used as experimental animals, weighing 20g ± 2g. Mice were acclimatized for 4–7 days before the experiment began.

[0222] S8.1: Mice were randomly divided into 7 groups at different time points (1d, 3d, 5d, 7d, 9d, 11d, 13d), with 5 mice in each group. Each mouse was injected twice a day for 4 consecutive days.

[0223] The dosage for each injection is as follows: PTTX1 2.5 mg / kg; PTTX2 0.28 mg / kg; PTTX3 0.20 mg / kg; PTTX4 0.15 mg / kg.

[0224] S8.2: At 1d, 3d, 5d, 7d, 9d, 11d and 13d after injection, tissue samples and blood samples were collected from the eyes of the five mice in the corresponding time group.

[0225] S8.3: Let the collected whole blood sample stand for 1 hour, then centrifuge at 3000 rpm for 15 minutes. Carefully aspirate the supernatant to obtain the serum sample, aliquot it into EP tubes, and store at -80℃ for later testing.

[0226] S8.4: Before testing, remove the serum samples and TTX ELISA kit from the refrigerator and allow them to return to room temperature. Use a tissue homogenizer to immerse and pulverize the remaining samples, centrifuge at 3000 rpm for 15 minutes, collect the supernatant, and aliquot it into EP tubes for testing. Then, prepare the required standards and washing buffer according to the kit instructions.

[0227] S8.5: Add TTX standards of various concentrations and the test sample (two replicates per sample) sequentially to the microplate, 15 μL per well. Immediately afterwards, add 50 μL of horseradish peroxidase (HRP)-labeled detection antibody, gently shake to mix, cover with a membrane, and incubate at 37°C for 60 minutes.

[0228] S8.6: Discard the liquid, fill each well with washing solution, let stand for 1 minute and then discard. Repeat the washing process 5 times, and pat dry on absorbent paper on the last wash.

[0229] S8.7: Add 50 μL of substrate A and substrate B to each well in sequence, and develop the color at 37°C in the dark for 15 minutes.

[0230] S8.8: Add 50 μL of stop solution to each well and immediately measure the absorbance (OD value) of each well at a wavelength of 450 nm using a microplate reader.

[0231] S8.9: Plot a standard curve with the standard concentration on the x-axis and the corresponding OD value on the y-axis. Substitute the OD values ​​of the samples into the standard curve to calculate the TTX concentration in each serum sample.

[0232] Table 14 Plasma Concentrations of PTTX Conjugate After Multiple Doses

[0233] Table 14 shows that the TTX blood concentration in mice injected with PTTX1 remained at a high level of approximately 50 pg / mL from day 1 to day 5, then slowly decreased, still remaining above 20 pg / mL at day 13. This indicates that after repeated administration of PTTX1, the drug significantly accumulates in the blood and is eliminated slowly. The TTX plasma concentrations in mice injected with PTTX2, PTTX3, and PTTX4 showed an overall continuous decreasing trend: PTTX2 decreased from 13.5 pg / mL at day 1 to 4.8 pg / mL at day 13, PTTX3 from 6.4 pg / mL to 2.2 pg / mL, and PTTX4 from 7.7 pg / mL to 2.0 pg / mL. The magnitude of the decrease in plasma concentration over time varied among the groups: the concentration decrease for PTTX1 (from day 1 to day 13) reached 52.0%, while that for PTTX4 reached 74.0%, suggesting that the elimination rates of different components in vivo may differ.

[0234] Experimental Example 9

[0235] Mice were used as experimental animals, weighing 20g ± 2g. After acclimatization for 4–7 days, the mice were used for experiments to assess their general physiological condition.

[0236] S9.1: Mice were randomly divided into groups of 5 mice at different time points. Each group of mice was intraperitoneally injected with the PTTX1 formulation prepared in Example 1 at a dose of 2.5 mg / kg body weight.

[0237] S9.4: On the day of administration (referred to as day 0), and on days 1, 2, 3... after administration (total number of days determined according to the experimental design), indicators shall be monitored at a fixed time each morning (e.g., 9:00-10:00).

[0238] S9.5: Weight monitoring: Using an electronic balance with an accuracy of 0.1g, weigh and record the weight of each mouse in sequence.

[0239] S9.6: Feed intake monitoring: Weigh and record the total weight of feed for each cage of mice daily, subtract the weight of the remaining feed for the next day, calculate the total daily feed intake of the mice in that cage, and then divide by the number of mice in the cage to obtain the average daily feed intake per mouse.

[0240] S9.7: Water intake monitoring: Record the total drinking water consumption of each cage of mice daily using a graduated cylinder or weighing method (weigh the water bottle filled with water and subtract the remaining weight the next day), and calculate the average daily water intake of each mouse.

[0241] S9.8: Observe and record the general condition of the mice daily, including their mental state, fur, activity behavior, and stool characteristics.

[0242] S9.9: Enter the daily monitoring data such as weight, food intake, and water intake into an electronic spreadsheet in a timely manner and perform statistical analysis.

[0243] S9.10: The control group followed the steps S9.1-S9.9 above, except that the intraperitoneal injection was replaced with physiological saline injection.

[0244] The experiment revealed that the food intake and water consumption of mice in both the PTTX1 injection group and the control group fluctuated during the experimental period (10 days), but there was no statistically significant difference between the two groups. The variation in food intake in both groups remained within 20% above and below baseline, indicating that PTTX1 injection did not significantly affect the feeding behavior of the mice. The weight changes in both groups showed similar trends, remaining within 5% of baseline; there were no significant differences in behavioral status between the two groups. The experimental results indicate that PTTX1 did not induce abnormal changes in mouse weight.

Claims

1. A tetrodocin coupling compound, characterized in that, The general formula of the tetrodocin conjugate is: Wherein, R is a synthetically produced carboxyl-containing polymer skeleton or a naturally sourced carboxyl-containing polymer skeleton, and the carboxyl-containing polymer is selected from carboxylated dextran, carboxylated β-cyclodextrin, alginate, or polyacrylic acid.

2. The tetrodocin conjugate as described in claim 1, characterized in that, The structural formula of the tetrodocin conjugate is selected from formulas (I), (II), (III), and (IV): In formula (I), n = 1 - 20000; In formula (II), R = COCH2CH2COOH or H. In formula (Ⅲ), m = 1 - 2000, n = 1 - 1900; In formula (Ⅳ), m = 1 - 9000 and n = 1 - 10000.

3. A method for preparing the tetrodocin conjugate according to any one of claims 1 or 2, characterized in that, The tetrodoxin conjugate is obtained by coupling reaction using a carboxyl-containing polymer and tetrodoxin as raw materials, with the addition of a catalyst and solvent; the carboxyl-containing polymer is selected from carboxylated dextran, carboxylated β-cyclodextrin, alginate, or polyacrylic acid.

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, and N,N′-dicyclohexylcarboimide, and is compounded with N,N-dimethylaminopyridine or other organic bases; the solvent for the coupling reaction is selected from non-hydroxyl solvents.

5. The preparation method according to claim 4, characterized in that, The solvent for the coupling reaction is selected from either dimethyl sulfoxide or N,N-dimethylformamide.

6. An application of a tetrodocin conjugate, characterized in that, The tetrodoxin conjugate according to any one of claims 1 or 2 is used to prepare a sodium channel blocker pharmaceutical composition.

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

8. The tetrodoxin conjugate formulation as described in claim 7, characterized in that, 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, physiological saline, acetic acid, gelatin, gum arabic, lactose, starch, cellulose, magnesium stearate, talc, vegetable oil, cyclodextrin, polyalkylene glycols, etc.

9. The tetrodoxin conjugate formulation as described in claim 7, characterized in that, The dosage form of the tetrodoxin conjugate is an injection.

10. The application of the tetrodocin conjugate as described in claim 6, characterized in that, The pharmaceutical composition is an injectable preparation.

11. An application of a tetrodocin coupling compound, characterized in that, The tetrodoxin conjugate according to any one of claims 1 or 2 is used to prepare a pharmaceutical composition for relieving pain.

12. An application of a tetrodocin conjugate, characterized in that, The tetrodoxin conjugate according to any one of claims 1 or 2 is used to prepare a pharmaceutical composition for relieving neuropathic pain.

13. The tetrodoxin conjugate formulation as described in claim 7, characterized in that, The tetrodoxin conjugate formulation is hydrolyzed and releases tetrodoxin upon entering the body; the drug window of the tetrodoxin conjugate formulation can be altered by selecting the molecular formula of the tetrodoxin conjugate and changing the type and / or quantity of the pharmaceutically acceptable excipients added.

Images