Preparation of tetrodotoxin external preparation and application of tetrodotoxin external preparation in treatment of postherpetic neuralgia
By using tuterotoxin as a topical preparation of Nav1.7 inhibitor, the problem of lack of drugs in the treatment of postherpetic neuralgia was solved, and the pain sensitivity and pain-related indicators of patients were significantly improved.
Patent Information
- Application Number
- CN202311788739.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-25
- Publication Date
- 2025-06-27
AI Technical Summary
The prior art lacks effective methods for treating postherpetic neuralgia, and there is a lack of clinical treatment drugs for this disease.
Trophotoxin was used as a Nav1.7 inhibitor, combined with polyethylene glycol-7-stearate (Tefose 63), carbomer 974 and sodium hydroxide solution, to prepare a topical preparation for the treatment of postherpetic neuralgia.
By inhibiting the Nav1.7 channel, the topical preparation of tuterotoxin significantly improves the mechanical and thermal pain sensitivity of patients with postherpetic neuralgia, and reduces the generation of pain-related biochemical indicators and inflammatory factors.
Smart Images
Figure CN120204232A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to an external preparation of tetrodotoxin, and specifically discloses a preparation method of an external preparation of tetrodotoxin and its application in the treatment of postherpetic neuralgia. Background Art
[0002] Postherpetic neuralgia is a common neuropathic pain caused by the reactivation of varicella-zoster virus latent in the dorsal root ganglion of the spinal cord or cranial nerve ganglia. The clinical manifestations of postherpetic neuralgia are burning and stabbing pain, with the characteristics of persistence and recurrence. Approximately 10% - 20% of patients infected with varicella-zoster virus suffer from postherpetic neuralgia. Currently, there are about 4 million patients with postherpetic neuralgia in China. There is a lack of clinical treatment drugs for postherpetic neuralgia, and there is currently no specific radical cure method for postherpetic neuralgia.
[0003] Voltage-gated sodium channel subtype Nav1.7 is a transmembrane channel protein that mediates the influx of sodium ions on the cell membrane. Nav1.7 is mainly distributed in peripheral nociceptive neurons, such as DRG and trigeminal ganglia, and can be activated by subthreshold potentials. It is the threshold channel for the generation of neuronal action potentials and mediates the conduction of nociception in the body. Genetic studies have shown that the gain-of-function mutation of the Nav1.7-encoding gene SCN9A causes hyperalgesia in patients with erythromelalgia, while loss-of-function mutations cause congenital insensitivity to pain. Nav1.7 has been proven to be a novel and valuable analgesic target. Clinical studies have shown that Nav1.7 is involved in the regulation of hyperalgesia in postherpetic neuralgia. Summary of the Invention
[0004] Based on the above background, the purpose of the present invention is to disclose a novel external preparation for the treatment of postherpetic neuralgia using tetrodotoxin, an inhibitor of Nav1.7, as the raw material drug, and a preparation method of the preparation.
[0005] The tetrodotoxin described in the present invention has a structure as shown in general formula (I) To achieve the above object, an external preparation for the treatment of postherpetic neuralgia proposed by the present invention is composed of the following materials: tetrodotoxin, polyethylene glycol-7-stearate (Tefose 63), carbomer 974, sodium hydroxide solution (0.5 mol / L), etc.
[0006] Furthermore, its composition includes tetrodotoxin at different concentrations.
[0007] The present invention also provides a preparation process of the novel external preparation for the treatment of postherpetic neuralgia, including the following steps: (1) Weigh purified water and Carbomer 974 in an appropriate ratio, swell for 10 - 20 h, and prepare a Carbomer solution. (2) Weigh Tefose 63 at 2 - 10% of the weight of the topical preparation, dissolve it into a clear liquid in a water bath at 60 - 80 °C, and use it as the oil phase. (3) Add the prescribed amount of tetrodotoxin to the oil phase in (2) above. (4) Under the condition of a water bath at 60 - 80 °C, slowly add the Carbomer solution in (1) above while stirring, and after adding, make up the remaining amount with purified water and stir evenly. (5) Take it out and continue to stir while cooling at room temperature. After cooling to room temperature, use sodium hydroxide solution to adjust the pH until the cream becomes a non - flowing semi - solid state. (6) Use a high - shear emulsifier for shearing until the cream is delicate and smooth. (7) Fill it into an aluminum tube and seal it.
[0008] Preferably, in step (1), the ratio of purified water to Carbomer 974 used is 49:1, and the swelling time is 12 - 15 h.
[0009] Preferably, in step (2), the amount of Tefose 63 used is 3 - 5% of the weight of the topical preparation.
[0010] Preferably, in step (5), the concentration of the sodium hydroxide solution used is 0.5 mol / L.
[0011] The present invention finally also provides the application of the said topical preparation in the preparation of a drug for treating post - herpetic neuralgia.
[0012] During application, evenly apply the said topical preparation to the skin of the affected area. Description of the Drawings
[0013] Figure 1 : TTX potently inhibits Nav1.7 channels. A, Tetrodotoxin inhibits Nav1.7 current. B, Dose - effect determination of tetrodotoxin inhibiting Nav1.7.
[0014] Figure 2 : Resiniferatoxin RTX potently activates TRPV1. A, Structure of resiniferatoxin; B, Mass spectrometry identification of resiniferatoxin; C, Resiniferatoxin activates TRPV1 channels; D, Dose - effect determination of resiniferatoxin activating TRPV1.
[0015] Figure 3 : TTX cream significantly improves mechanical hyperalgesia induced by resiniferatoxin RTX. In a rat model of post - herpetic neuralgia induced by resiniferatoxin RTX, time - course changes of mechanical pain thresholds before and after administration of tetrodotoxin topical cream.
[0016] Figure 4 : The TTX cream effectively improves the thermal pain threshold in rats induced by resiniferatoxin (RTX). The time-course changes of the thermal pain threshold in a rat model of postherpetic neuralgia induced by RTX before and after the application of the tetrodotoxin topical cream are presented.
[0017] Figure 5 : The TTX cream effectively inhibits the generation of pain stress factors in the serum of rats with postherpetic neuralgia induced by RTX. A. The tetrodotoxin topical cream significantly inhibits the generation of cortisol in the serum; B. The tetrodotoxin topical cream significantly inhibits the generation of norepinephrine in the serum; C. The tetrodotoxin topical cream significantly inhibits the generation of substance P in the serum.
[0018] Figure 6 : The TTX cream effectively inhibits the expression of pain-related inflammatory factors in rats with postherpetic neuralgia induced by RTX. A. The tetrodotoxin topical cream significantly inhibits the generation of TNF-α in the spinal cord tissue; B. The tetrodotoxin topical cream significantly inhibits the generation of IL-6 in the spinal cord tissue.
[0019] Figure 7 : General formula (I). Embodiment
[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It should be noted that the preparation methods of the following embodiments are as described above and will not be repeated one by one. Only the functional components of the topical preparation will be described.
[0021] Example 1 The functional components of the topical preparation in the example: 0.005‰ tetrodotoxin.
[0022] Example 2 The functional components of the topical preparation in this example: 0.05‰ tetrodotoxin.
[0023] Example 3 The functional components of the topical preparation in this example: 0.5‰ tetrodotoxin.
[0024] Example 4 The functional components of the topical preparation in this example: 1.5‰ tetrodotoxin.
[0025] Comparative Example 1 The topical preparation in this comparative example: compound lidocaine cream The invention will be further described below with reference to the accompanying drawings.
[0026] Experimental Example: Rat Animal Model The present invention uses patch clamp technology to verify that tetrodotoxin potently inhibits the pain target voltage-gated sodium channel subtype Nav1.7. The results showed that tetrodotoxin inhibited Nav1.7 peak current in a concentration-dependent manner, IC 50 The value is 9.82 nM ( Figure 1 ).
[0027] The activation of TRPV1 channel by resiniferatoxin (RTX) was measured using a rapid fluorescence imaging analyzer.
[0028] Resiniferatoxin is a diterpenoid compound extracted from the resin of Euphorbia pulegiana, which has a potent agonist activity against the capsaicin receptor TRPV1 ( Figure 2 A). The molecular weight of RTX was identified and the TRPV1 agonist activity was evaluated. The results are shown in Figure 4B. The molecular weight of RTX was determined to be 628.71, which is consistent with the theoretical value ( Figure 2 B). Real-time fluorescent calcium imaging showed that compound RTX could rapidly induce [Ca 2+ ] i The increase ( Figure 2 C). RTX can enhance the TRPV1-mediated intracellular [Ca 2+ ] i Increase, determine EC 50 is 137 nM, which is consistent with the literature report ( Figure 2 D).
[0029] (1) Experimental animals SD rats (purchased from the Medical Experimental Animal Center of Nanjing Medical University, The rats were kept in an SPF animal room with a light-to-dark ratio of 1:1, room temperature at 23 ± 2°C, humidity at 55%, and free access to food and water. The litter was changed every 3 days to ensure that the rats were in a dry and clean environment. All animal experimental protocols were approved by the Laboratory Animal Care and Use Committee of China Pharmaceutical University (License No.: SYXK (Su) 2018-0019).
[0030] (2) Experimental drugs and instruments Tetrodotoxin (TTX), manufacturer: Zhongyang Biotechnology (Jiangsu) Co., Ltd. Resiniferatoxin (RTX), manufacturer: CFWLABS, batch number: A-125100-NU3.
[0031] Compound Lidocaine Cream, Manufacturer: Tongfang Pharmaceutical Group Co., Ltd. (3)Construction of animal model of postherpetic neuralgia After 80 SPF-grade SD rats, 4 - 6 weeks old and weighing 150 - 200 g, were adaptively fed for 3 days, they were anesthetized by intraperitoneal injection of 2% sodium pentobarbital and then intraperitoneally injected with resiniferatoxin (200 μg / kg) once. The successful construction of the model was based on the criteria of reduced mechanical withdrawal threshold and prolonged thermal withdrawal latency in rats. Animal experiments followed the Guidelines for the Breeding, Management and Use of Laboratory Animals of China Pharmaceutical University and conformed to animal ethics specifications.
[0033] (4)Administration route and dosage 50 mg of blank cream, 0.005‰, 0.05‰, 0.5‰, 1.5‰ TTX cream and compound lidocaine cream were weighed separately with an electronic analytical balance and evenly applied to the surface of the right hind paw of SD rats once a day. (5)Group classification and drugs applied to each group are as follows: Blank control group (Control): No treatment; Model group (Model): Induced by RTX injection; Model + vehicle group: Induced by RTX injection; Applied with blank cream without TTX; Positive drug group: Induced by RTX injection; Applied with compound lidocaine cream; Low-dose TTX group (0.005‰): Induced by RTX injection; Applied with 0.005‰ TTX cream; Low-dose TTX group (0.05‰): Induced by RTX injection; Applied with 0.05‰ TTX cream; Medium-dose TTX group (0.5‰): Induced by RTX injection; Applied with 0.5‰ TTX cream; High-dose TTX group (1.5‰): Induced by RTX injection; Applied with 1.5‰ TTX cream; (6)Von Frey method Experimental rats were placed in an experimental box sized 30 cm×30 cm×30 cm. After 30 min of adaptation, the mechanical pain threshold was measured using Von Frey. The nylon filament was vertically stimulated on the plantar skin of the right hind paw of the rat in a quiet state. The force was gradually increased to bend the filament into an angle, lasting for 1.5 s. It was continuously detected 5 times, and the detection interval was more than 5 min to let the rat calm down. When the rat showed any behavior such as hissing, licking the paw, tail flicking, or leg kicking, it was recorded as positive for that force. If ≥3 times were positive in the 5 determinations, it was recorded as positive. In case of a positive reaction, the next smaller-sized filament was selected to continue the stimulation until the stimulation with the smaller-sized filament was negative. If it was negative, a filament with a greater strength was selected until a positive reaction occurred.
[0034] The results showed that after modeling by intraperitoneal injection of RTX, the mechanical paw withdrawal threshold of each modeling group was significantly decreased and tended to be stable on the fourth day ( Figure 3 ). On the 5th day, different doses of TTX cream and compound lidocaine cream were given respectively. TTX creams at doses of 0.005‰ and 0.05‰ could significantly improve mechanical hyperalgesia, and were stronger than the positive control drug compound lidocaine cream. As the dose was further increased, TTX creams at 0.5‰ and 1.5‰ could also improve mechanical hyperalgesia.
[0035] (7) Hot plate method SD rats were subjected to adaptive training for heat pain stimulation testing using a rat automatic hot plate apparatus, and rats with abnormal heat pain were excluded. The temperature of the automatic hot plate apparatus was set at 30°C, and the paw withdrawal latency of the rat was measured (recording the time from when the four limbs of the rat touched the hot plate until the rat retracted its hind paw and licked its hind paw), which was the heat stimulation response latency. It was measured 4 times repeatedly, with an interval of 5 min each time.
[0036] The results showed that after RTX modeling, the heat pain sensitivity remained at a low level, and the heat pain sensitivity of the drug treatment group was restored to varying degrees; TTX cream at 0.005‰ could also improve the insensitivity of the heat pain threshold. As the dose increased, TTX creams at 0.05‰, 0.5‰, and 1.5‰ could all restore the heat pain sensation of the model rats, showing the same improvement effect as the positive drug compound lidocaine cream ( Figure 4 )
[0037] Detection of pain stress biochemical indexes On the 6th day of TTX treatment, blood was taken from the orbital cavities of rats in each group, and the supernatant was obtained by centrifugation. An ELISA kit was used to detect the pain stress indexes of the body, namely the changes in cortisol, norepinephrine, and substance P in serum.
[0038] The results of serum cortisol content determination showed that the cortisol content in the control group was 122.70 ± 2.46 ng / mL, which increased to 131.30 ± 3.03 ng / mL after RTX modeling. After drug treatment, the cortisol content in the compound lidocaine group was 112.00 ± 5.83 ng / mL, and the serum cortisol contents in the 0.005‰, 0.05‰, 0.5‰ and 1.5‰ TTX cream groups were 119.60 ± 4.88 ng / mL, 115.90 ± 4.88 ng / mL, 114.60 ± 2.44 ng / mL, 113.70 ± 3.71 ng / mL respectively. Compared with the modeling group, the 4-dose TTX topical cream could significantly improve the secretion of serum cortisol ( Figure 5 A) (P<0.01).
[0039] The results of serum norepinephrine content determination showed that the norepinephrine content in the control group was 3.45 ± 0.58 ng / mL, which increased to 4.24 ± 0.59 ng / mL after RTX modeling. After drug administration, the norepinephrine content in the compound lidocaine group was 2.05 ± 0.21 ng / mL, and the serum norepinephrine contents in the 0.005‰, 0.05‰, 0.5‰ and 1.5‰ TTX cream groups were 2.75 ± 0.37 ng / mL, 2.12 ± 0.22 ng / mL, 2.20 ± 0.35 ng / mL, 2.23 ± 0.27 ng / mL respectively. Compared with the modeling group, the 4-dose TTX topical cream could significantly improve the secretion of serum norepinephrine ( Figure 5 B) (P<0.01).
[0040] The results of serum substance P content determination showed that the substance P content in the control group was 6.77 ± 0.34 ng / mL, which increased to 7.52 ± 0.77 ng / mL after RTX modeling. After drug administration, the substance P content in the compound lidocaine group was 4.44 ± 0.46 ng / mL, and the serum substance P contents in the 0.005‰, 0.05‰, 0.5‰ and 1.5‰ TTX cream groups were 3.51 ± 0.16 ng / mL, 3.63 ± 0.25 ng / mL, 5.05 ± 0.26 ng / mL, 3.56 ± 0.49 ng / mL respectively. Compared with the modeling group, the 4-dose TTX topical cream could significantly improve the secretion of serum substance P ( Figure 5 C) (P<0.05).
[0041] (9)Detection of pain-related inflammatory factors On the 6th day of TTX treatment, rats in each group were anesthetized by intraperitoneal injection of 2% sodium pentobarbital. The spinal cord segments L4-L6 were taken, homogenized by ultrasound in an ice bath environment, and the supernatant was extracted after centrifugation. ELISA kits were used to detect the changes in pain-related inflammatory factors TNF-α and IL-6 respectively.
[0042] The results of the determination of TNF-α content showed that the TNF-α content in the control group was 245.60 ± 17.98 pg / mL, and it increased to 276.50 ± 19.59 pg / mL after RTX modeling. After drug treatment, the IL-6 content in the compound lidocaine group was 209.7 ± 17.35 ng / mL, and the TNF-α contents in the 0.005‰, 0.05‰, 0.5‰ and 1.5‰ TTX cream groups were 259.50 ± 7.98 pg / mL, 201.00 ± 14.17 pg / mL, 197.50 ± 22.37 pg / mL, 164.70 ± 21.71 pg / mL respectively. Compared with the modeling group, the 0.05‰, 0.5‰ and 1.5‰ TTX cream groups could significantly improve the production of TNF-α ( Figure 6 A)(P<0.05).
[0043] The results of the determination of IL-6 content showed that the content in the control group was 117.20 ± 4.11 pg / mL, and it increased to 128.1 ± 7.68 pg / mL after RTX modeling. After drug administration, the IL-6 content in the compound lidocaine group was 105.0 ± 4.47 pg / mL, and the serum IL-6 contents in the 0.005‰, 0.05‰, 0.5‰ and 1.5‰ TTX cream groups were 86.76 ± 5.54 pg / mL, 73.45 ± 2.77 pg / mL, 100.9 ± 5.90 pg / mL, 85.54 ± 2.89 pg / mL respectively. Compared with the modeling group, the TTX topical cream at doses of 0.005‰, 0.05‰ and 1.5‰ could significantly improve the secretion of serum IL-6 ( Figure 6 B)(P<0.05).
[0044] Responsible for Figures 3 to 6 TTX cream significantly alleviated the pain sensitivity of mechanical pain in the modeled rats. At the same time, TTX cream effectively improved the thermal pain threshold of the modeled rats. The determination of rat serum biochemical indexes showed that TTX cream effectively inhibited the production of pain stress factors in the serum, and at the same time, TTX cream effectively inhibited the expression of pain-related inflammatory factors in the spinal cord tissue.
Claims
1. Preparation of a tetrodotoxin topical preparation and its application in the treatment of postherpetic neuralgia, characterized in that, The method comprises the following steps: (1) The topical preparation is mainly made of tetrodotoxin at different concentrations, and topical preparations with tetrodotoxin contents of 0.005‰, 0.05‰, 0.5‰, and 1.5‰ are prepared. (2) An animal model of postherpetic neuralgia is established. (3) Animal experiments are conducted to study the application of the tetrodotoxin topical preparation in drugs for treating postherpetic neuralgia.
2. The preparation of a tetrodotoxin external preparation as described in claim 1 and its application in the treatment of postherpetic neuralgia, characterized in that, In step (1), the preparation method of the topical preparation is as follows: tetrodotoxin is added to the oil phase made of polyethylene glycol-7-stearate (Tefose 63) and mixed evenly, then the aqueous phase made of carbomer 974 is added to the oil phase, purified water is added to make up the full amount, and the mixture is stirred evenly; while cooling at room temperature, stirring is continued; after cooling to room temperature, the pH is adjusted to a non-flowable semi-solid state of the cream using sodium hydroxide solution. Through processes such as shearing, filling, and sealing, the topical preparation is obtained.
3. The preparation of a tetrodotoxin topical preparation as described in claim 1 and its application in the treatment of postherpetic neuralgia, characterized in that, In step (2), the animals used to establish the animal model of postherpetic neuralgia are SD rats.
4. The preparation of a tetrodotoxin external preparation as described in claim 1 and its application in the treatment of postherpetic neuralgia, characterized in that, In step (2), the method used to establish the animal model of postherpetic neuralgia is to intraperitoneally inject resiniferatoxin into SD rats.
5. The preparation of an external preparation of tetrodotoxin as claimed in claim 1 and its application in the treatment of postherpetic neuralgia, characterized in that, In step (3), the methods used in the animal experiments to study the application of the tetrodotoxin topical preparation in drugs for treating postherpetic neuralgia include the von Frey method, the hot plate method, the detection of pain stress biochemical indicators, and the detection of pain-related inflammatory factors, etc.