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Volume 41 Issue 9
Sep.  2023
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XU Jiren, ZHENG Ziyun, LI Yuan, LU Ying, CHU Zhiyong. Preliminary pre-prescription study of Tetrodotoxin[J]. Journal of Pharmaceutical Practice and Service, 2023, 41(9): 544-546, 551. doi: 10.12206/j.issn.2097-2024.202304024
Citation: XU Jiren, ZHENG Ziyun, LI Yuan, LU Ying, CHU Zhiyong. Preliminary pre-prescription study of Tetrodotoxin[J]. Journal of Pharmaceutical Practice and Service, 2023, 41(9): 544-546, 551. doi: 10.12206/j.issn.2097-2024.202304024

Preliminary pre-prescription study of Tetrodotoxin

doi: 10.12206/j.issn.2097-2024.202304024
  • Received Date: 2023-04-14
  • Rev Recd Date: 2023-08-14
  • Available Online: 2023-09-25
  • Publish Date: 2023-09-25
  •   Objective   To investigate the solubility and stability of Tetrodotoxin (TTX) in different solvents, and the effect of temperature and pH on its stability.   Method   Solutions of TTX in different matrices were prepared. Their concentrations at different temperatures and pH buffers were determined by high performance liquid chromatography and their solubility and stability were analyzed and calculated.   Result  TTX was most soluble at pH 3.5 and its solubility decreased as the pH increased. TTX degraded most rapidly under strong alkali conditions, with complete degradation after 20 min of reaction at 0.1 mol/L sodium hydroxide and 70 ℃. The stability test results similarly demonstrated that TTX was least stable under alkaline conditions. In a PBS buffered solution at 37 ℃, pH 7.4, TTX concentration began to decrease consistently at 1~10h, with a degradation rate of 88.07±0.27% after 28 days.   Conclusion   TTX is readily soluble in acidic aqueous solutions at pH 3.5 and almost insoluble in alkaline aqueous solutions. Its stability is closely related to the temperature and pH of the medium. It is more stable in acidic aqueous solutions and easily degrades under alkaline conditions, and its degradation process could be accelerated by increasing temperature.
  • [1] 方国锋, 王锡昌, 陶宁萍, 等. 河豚毒素的样品前处理与快速检测技术研究进展[J]. 分析测试学报, 2014, 33(12): 1447-1452. doi:  10.3969/j.issn.1004-4957.2014.12.021
    [2] BANE V, LEHANE M, DIKSHIT M, et al. Tetrodotoxin: chemistry, toxicity, source, distribution and detection[J]. Toxins, 2014, 6(2): 693-755. doi:  10.3390/toxins6020693
    [3] SAOUDI M, ABDELMOULEH A, EL FEKI A. Tetrodotoxin: a potent marine toxin[J]. Toxin Rev, 2010, 29(2): 60-70. doi:  10.3109/15569543.2010.487631
    [4] NOGUCHI T. Tetrodotoxin - distribution and accumulation in aquatic organisms, and cases of human intoxication[J]. Mar Drugs, 2008, 6(2): 220-242. doi:  10.3390/md6020220
    [5] 张梦倩, 储智勇, 钱晓明等. 河鲀毒素——一种具有缓解癌性疼痛潜力的药物[J]. 生物化学与生物物理进展, 2023, 50(7): 1-15.
    [6] SHI J, LIU T T, WANG X, et al. Tetrodotoxin reduces cue-induced drug craving and anxiety in abstinent heroin addicts[J]. Pharmacol Biochem Behav, 2009, 92(4): 603-607. doi:  10.1016/j.pbb.2009.02.013
    [7] 王兴龙, 蔡强, 桂文锋, 等. 河鲀毒素及其检测技术研究进展[J]. 水产科学, 2020, 39(3): 447-457.
    [8] KATIKOU P, GOKBULUT C, KOSKER A R, et al. An updated review of tetrodotoxin and its peculiarities[J]. Mar Drugs, 2022, 20(1): 47. doi:  10.3390/md20010047
    [9] 张金艳, 姜雯鹏, 谭欣, 等. 基于AuPtRh纳米酶的比色适体传感器快速检测河豚毒素[J]. 食品安全质量检测学报, 2022, 13(22): 7183-7190.
    [10] 国家药典委员会. 中华人民共和国药典2020年版. 四部[S]. 北京: 中国医药科技出版社, 2020, 424-425.
    [11] GOLDLUST S A, KAVOOSI M, NEZZER J, et al. Tetrodotoxin for chemotherapy-induced neuropathic pain: a randomized, double-blind, placebo-controlled, parallel-dose finding trial[J]. Toxins, 2021, 13(4): 235. doi:  10.3390/toxins13040235
    [12] CAMPOS-RÍOS A, RUEDA-RUZAFA L, HERRERA-PÉREZ S, et al. Tetrodotoxin: a new strategy to treat visceral pain?[J]. Toxins, 2021, 13(7): 496. doi:  10.3390/toxins13070496
    [13] BUCCIARELLI G M, LECHNER M, FONTES A, et al. From poison to promise: the evolution of tetrodotoxin and its potential as a therapeutic[J]. Toxins, 2021, 13(8): 517. doi:  10.3390/toxins13080517
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Preliminary pre-prescription study of Tetrodotoxin

doi: 10.12206/j.issn.2097-2024.202304024

Abstract:   Objective   To investigate the solubility and stability of Tetrodotoxin (TTX) in different solvents, and the effect of temperature and pH on its stability.   Method   Solutions of TTX in different matrices were prepared. Their concentrations at different temperatures and pH buffers were determined by high performance liquid chromatography and their solubility and stability were analyzed and calculated.   Result  TTX was most soluble at pH 3.5 and its solubility decreased as the pH increased. TTX degraded most rapidly under strong alkali conditions, with complete degradation after 20 min of reaction at 0.1 mol/L sodium hydroxide and 70 ℃. The stability test results similarly demonstrated that TTX was least stable under alkaline conditions. In a PBS buffered solution at 37 ℃, pH 7.4, TTX concentration began to decrease consistently at 1~10h, with a degradation rate of 88.07±0.27% after 28 days.   Conclusion   TTX is readily soluble in acidic aqueous solutions at pH 3.5 and almost insoluble in alkaline aqueous solutions. Its stability is closely related to the temperature and pH of the medium. It is more stable in acidic aqueous solutions and easily degrades under alkaline conditions, and its degradation process could be accelerated by increasing temperature.

XU Jiren, ZHENG Ziyun, LI Yuan, LU Ying, CHU Zhiyong. Preliminary pre-prescription study of Tetrodotoxin[J]. Journal of Pharmaceutical Practice and Service, 2023, 41(9): 544-546, 551. doi: 10.12206/j.issn.2097-2024.202304024
Citation: XU Jiren, ZHENG Ziyun, LI Yuan, LU Ying, CHU Zhiyong. Preliminary pre-prescription study of Tetrodotoxin[J]. Journal of Pharmaceutical Practice and Service, 2023, 41(9): 544-546, 551. doi: 10.12206/j.issn.2097-2024.202304024
  • 河豚毒素(TTX)属于氨基全氢化喹唑啉化合物,分子式为C11H17O8N3,分子量为319。其分子结构类似于生物碱[1],呈独特的笼形,如图1。河豚毒素粗品为黄褐色粉末,纯品为白色晶体,呈弱碱性,容易潮解,微溶于水溶液,易溶于无机酸水溶液,且在弱酸条件下稳定;对于热不敏感,但在高温下毒性会增强,强酸强碱条件下其结构可被破坏[2]

    河豚毒素是一种高效的神经类毒素,由于能够特异性的阻断Na+离子通道,进而产生麻痹作用,因此低浓度的河豚毒素被认为是一种优良的神经类候选药物,一直受到科研人员尤其是新药研发人员的关注[3]。目前已有将河豚毒素作为止痛药[4-5]和成瘾戒断药[6]的相关探索和研究。由于河豚毒素在药学领域有着广阔前景,关于其药学应用的研究报道逐年增多,但大多集中在分析方法[7]、药理机制[8]或药物载体研究[9],对其可成药性的相关研究报道较少。本文按照新药研发要求,建立高效液相反相离子对色谱法,对河豚毒素在不同溶剂中的溶解性和不同pH环境中的稳定性进行考察,为河豚毒素的处方前研究积累数据,为其进一步临床应用提供有力支撑。

    • LC-2030C高效液相色谱仪(日本岛津制作所);AL-104电子天平(METTLER TOLEDO,瑞士);数显pH计(sartorius,德国);紫外可见分光光度计(安捷伦科技(中国)有限公司);循环水式真空泵(上海豫康科教仪器设备有限公司);SECURA125-1CN型十万分之一电子天平(赛多利斯,德国);Arium@ mini 超纯水机(赛多利斯,德国)。

    • 河豚毒素原料药(中洋生物科技江苏有限公司,批号:20220089,含量88%);河豚毒素对照品(泰州康特生物工程有限公司,批号:201206,含量≥99%);磷酸、98%甲酸、柠檬酸、磷酸二氢钠、磷酸氢二钠、磷酸二氢钾、磷酸氢二钾、氢氧化钠、庚烷磺酸钠、磷酸二氢铵、乙酸铵、盐酸、30% H2O2和冰醋酸均为分析纯试剂(国药集团化学试剂有限公司)。

    • 色谱柱为Shim-pack GISI C18-AQ(4.6×250 mm,5 μm);流动相为5 mmol/L庚烷磺酸钠-25 mmol/L磷酸二氢铵,等度洗脱;进样量为10 μl;柱温:25 ℃;流速1 ml/min;检测波长为196 nm;检测时长为40 min。

    • 取TTX对照品适量,精密称定,用0.1%磷酸溶液溶解并稀释制成浓度为1.0 mg/ml的TTX对照品储备液。

    • 取TTX原料药适量,精密称定,用0.1%磷酸溶液溶解并稀释制成浓度为1.0 mg/ml的供试品储备液。

    • 按照《中国药典》(2020版)四部通则8004缓冲液配制方法,配制pH值分别为3.5、4.5、5.0、6.5、7.0、7.4、8.0的缓冲液[10]。称取研成细粉的TTX原料药各2 mg,分别置于4 ml不同pH值的缓冲液(25±2 ℃)中,30 min内每隔5 min强力振摇30 s,观察溶解情况,如无目视可见颗粒,视为完全溶解。如完全溶解则继续加入研成细粉的TTX原料药,同法操作直至饱和,取适量经滤膜滤过,作为溶解度试验测试溶液。

    • 取研成细粉的TTX原料药约1 mg,置于10 ml容量瓶中,分别用0.1 mol/L盐酸和0.1 mol/L氢氧化钠溶液溶解并定容,置于70 ℃下反应20 min。精密量取各反应溶液适量,冷却至室温,分别加入对应体积的0.1 mol/L氢氧化钠或0.1 mol/L盐酸进行中和并终止反应。取适量样品经滤膜过滤后作为强酸与强碱破坏试验测试溶液。

    • 取研成细粉的TTX原料药约1 mg,置于10 ml容量瓶中,加入30% H2O2溶液溶解定容后,室温反应10 min。精密量取反应溶液适量,经滤膜过滤,作为氧化破坏试验测试溶液。

    • 取研成细粉的TTX各适量,精密称定,分别用PBS缓冲液(pH值=7.4)、0.1%甲酸溶液、0.1%柠檬酸溶液溶解,并稀释制成浓度为0.1 mg/ml的TTX溶液,然后分别置于4 ℃、25 ℃、37 ℃中储存,在第1、2、3、5、7、14、21、28 d各取适量经滤膜过滤,作为温度适应性测试溶液。

    • 分别将对照品储备液及供试品储备液用0.1%磷酸溶液稀释至0.1 mg/ml,作为对照品溶液和供试品溶液,按“2.1”项进行测试。如图2所示,TTX的保留时间为22.665 min。理论板数为14 501,供试品溶液中主峰与邻峰的分离度为6.4。结果表明,此色谱条件可以满足TTX相关测试要求。

    • 取对照品储备液,用0.1%磷酸溶液配制浓度为4、10、20、40、80 μg/ml的TTX对照品测试溶液,按“2.1”项进行测试。以浓度为横坐标(X),峰面积为纵坐标(Y),绘制标准曲线,得回归方程为Y=12 601.2X–2 263.69(r=0.999 9)。结果表明,TTX的浓度与峰面积在测试范围内呈良好线性关系。

    • 取对照品储备液,用0.1%磷酸溶液分别稀释至10、20、40 μg/ml的溶液各3份,按“2.1”项进行测试,计算其含量的平均值为101.000%,RSD为0.60%,表明该方法准确度良好,满足测试要求。

    • 取供试品溶液,按“2.1”项重复测试6次,计算得到的RSD为0.17%,表明该方法重复性良好。室温分别放置0、2、4、6、8、10、12、24、48 h后按“2.1”进行测试。计算得到的RSD为1.48%,表明供试品溶液在48 h内稳定性良好。

    • 表1结果可看出,TTX几乎不溶于碱性水溶液,但溶解度随着pH值降低而逐渐增大,易溶于pH值为3.5的酸性水溶液。

      pH值 3.5 4.5 5.0 6.5 7.0 7.4 8.0
      $ \bar{\rm{x}} $(mg/ml) 264.63 31.35 4.64 1.02 0.47 0.21 0.05
      SD(mg/ml) 5.54 0.75 0.08 0.046 0.015 0.002 7 0.001
    • 实验结果表明,强碱对TTX的破坏能力最强,在0.1mol/L氢氧化钠溶液中、70 ℃条件下反应20 min后,TTX被完全降解。强酸对TTX的破坏能力次之,TTX在70 ℃、0.1mol/L HCl溶液中反应20 min后的降解率为60.78%;氧化对TTX的破坏能力相对最弱,在30% H2O2溶液中反应20 min后的降解率为17.06%。

    • 不同介质、不同温度条件下TTX稳定性试验结果见图3。当溶剂为pH值=7.4的PBS溶液时,不同温度下TTX均表现出降解趋势,在25 ℃与37 ℃条件下保存28 d的TTX溶液降解率均超过50%,在37 ℃时PBS溶液中的TTX降解率更高达88.07±0.27%,提示在后续剂型研究中,需注意TTX在体外释放液中的稳定性。以0.1%甲酸溶液或0.1%柠檬酸溶液为溶剂的TTX溶液则较为稳定,在25 ℃和37 ℃下保存28 d含量均未表现出显著性变化。

    • TTX作为一种具有广泛应用前景的潜在镇痛药物,其在药理和作用机制方面的相关研究已有较多报道[11-13],但对于其理化性质和成药性研究的报道极少。本研究建立了高效液相反相离子对色谱法,测定TTX在不同溶剂中的溶解性,并考察温度、溶剂pH值和氧化剂对其稳定性的影响。实验结果表明TTX在酸性条件下具有较高的溶解度,前期试验还表明TTX不溶于乙醇,二氯甲烷等有机溶剂。稳定性试验结果显示TTX在强碱溶液中较不稳定,实验条件下20 min即被强碱全部降解,而在酸性条件、氧化环境中较为稳定。不同温度稳定性试验结果同样显示,TTX在0.1%甲酸或柠檬酸溶液中4周内稳定性均良好;而在pH值=7.4 PBS中降解较为迅速,即使在4 ℃条件下,pH值=7.4 PBS中28 d的降解率仍然接近50%,37 ℃条件下28 d降解率接近90%。研究结果可为TTX处方设计及制备工艺提供实验依据,并提示研究者在开展TTX剂型研究和体外释放研究时应重点关注其稳定性和降解情况。

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