WO2012126344A1 - 改性眼镜蛇毒及其制备方法和用途 - Google Patents

改性眼镜蛇毒及其制备方法和用途 Download PDF

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WO2012126344A1
WO2012126344A1 PCT/CN2012/072529 CN2012072529W WO2012126344A1 WO 2012126344 A1 WO2012126344 A1 WO 2012126344A1 CN 2012072529 W CN2012072529 W CN 2012072529W WO 2012126344 A1 WO2012126344 A1 WO 2012126344A1
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venom
cobra venom
modified
cobra
entire entire
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PCT/CN2012/072529
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English (en)
French (fr)
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秦正红
祝佳丽
王进
史高娜
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苏州大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/58—Reptiles
    • A61K35/583—Snakes; Lizards, e.g. chameleons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection

Definitions

  • the invention belongs to the technical field of drug preparation, and particularly relates to a physical modification method for modifying cobra venom and cobra venom and the use thereof in preparing oral, external and injectable drugs. Background technique
  • Snake venom is a complex mixture of various components secreted by the snake's venom glands. More than 90% of the dry matter of snake venom is protein, which is the main component of its toxicity and its biological activity. The traditional medicine of the motherland believes that the mirror snake and its toxic components can pass through the meridians, rheumatism, and have the effect of strengthening the body. As early as the beginning of the 20th century, people began to use snake venom to relieve malignant tumor pain, neuralgia and joint pain. Subsequently, a series of reports on the similar analgesic effects of snake venom have been reported.
  • the object of the present invention is to provide a modified cobra venom and to provide a novel analgesic drug.
  • the technical solution provided by the present invention is:
  • a modified cobra venom characterized in that the cobra venom is from Chinese cobra venom or Thai cobra venom, and the modified cobra venom is obtained by heating the cobra venom 60-100 ° C for 5-120 minutes and then cooling. Characterized small molecular weight protein when modified cobra venom is detected by SDS-PAGE increase. It has been confirmed by experiments that the modified cobra venom has enhanced drug efficacy and decreased toxicity.
  • Another object of the present invention is to provide a composition comprising modified cobra venom, characterized in that said composition comprises, by weight percent of its components:
  • a pharmaceutically acceptable carrier is 90 to 99.99 by weight.
  • the dosage form of the composition is selected from an oral dosage form or an external dosage form or an injection.
  • the oral dosage form is selected from the group consisting of an oral liquid, a capsule, and an oral drug;
  • the external dosage form is selected from the group consisting of a coating agent, a cataplasm, and a spray, and the injection dosage form is selected from the group consisting of a powder injection and a water injection.
  • a further object of the present invention is to provide a method for modifying a modified cobra venom, characterized in that the modification method is a physical modification method, which comprises heating the cobra venom at 60-100 ° C for 5-120 minutes. The step of cooling.
  • the cobra venom is dissolved in 0.5 M PBS buffer, and the pH of the solution is adjusted to be about 7.8.
  • the present invention relates to a physical modification method for venom venom and use in the preparation of oral, injectable and topical medicines.
  • the cobra venom of the present invention is subjected to heat treatment and other auxiliary conditions to change the content of some components to achieve synergistic effect;
  • the modified snake venom drug includes oral liquid, capsule, film, paint, cataplasm, spray and Various dosage forms such as injections.
  • the cobra venom of the present invention is derived from the Chinese cobra venom (Naja naja atra ) and the Thai venom venom
  • Cobra venom can be obtained by the following methods: collecting venom from captive Chinese or Thai scorpion snails, or purchasing cobra lyx lyophilized powder from the market.
  • the venom of the venom includes the snake venom produced by the Chinese cobra and the Thai cobra.
  • the medicament of the present invention also includes various unilateral and combination preparations.
  • Chinese cobra venom has been used clinically to treat rheumatism and relieve malignant tumor pain, neuralgia and joint pain, there are no reports in the literature about the total poison of Naja naja atra and the Thai Mirror Python snake. Naja Kaouthia can reduce toxicity and increase efficiency after heat treatment. It has also been reported that this modified snake venom can be clinically administered by oral, nasal (oral) mucosal administration, dermal administration, and injection administration.
  • the cobra venom solution is in 0.5 M PBS (pH 7.8), Heat at 60-100 ° C for about 120 minutes (range 5-120 minutes), quickly heat the cobra venom to cool in ice (or gradually cool).
  • the snake venom after this heat treatment showed an increase in characteristic small molecular weight proteins when detected by SDS-PAGE.
  • the analgesic effect is more effective and long-lasting, but also has better anti-inflammatory and immunomodulatory effects.
  • the venom of the venom of the present invention (including Chinese scorpion venom venom and Thai cobra venom) has a small amount of physical modification, and has a large effective range, and has good analgesic and immunomodulatory effects from lO-lOOOO g/kg. Role, the scope of security expanded.
  • the cobra venom of the present invention (including Chinese scorpion venom and Thai scorpion venom) has been physically modified and administered in a variety of routes including dermal administration, oral administration, oral (nasal) adhesion and injection.
  • the present invention discloses a method for physically modifying Naja naja atra, which has a physical modification of the venom of the venom of the venom, which is enhanced in analgesic and anti-inflammatory and immunosuppressive immunity.
  • the effect is more durable.
  • the venom of the venom is taken from the venom of Chinese or Thai scorpion snakes that have been artificially reared.
  • the research of the invention finds that the physical modification of the target venom venom (10-100Og/kg) has obvious analgesic and anti-inflammatory and immunosuppressive effects, and has small dosage, safety, oral administration, injectability and permeation. It has the advantages of oral and nasal mucosa and skin administration, and has great clinical practical value.
  • FIG. 1 is a diagram showing changes in protein components after heat treatment of cobra venom according to an embodiment of the present invention
  • FIG. 2 is a comparison diagram of the pain threshold of physical modification and modification before the target of 30 g/kg of venom of the venom of the present invention; wherein, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001 and administration The front pain threshold is compared.
  • FIG. 3 is a comparison diagram of the pain threshold of physical modification and modification before the target venom of 100 ng/kg of the venom of the present invention. detailed description
  • the invention discloses a physical modification method for modifying venom venom and cobra venom and the use thereof in preparing medicines for oral administration, external use and injection, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the products, methods, and applications of the present invention have been described by the preferred embodiments, and it is obvious that the methods and applications described herein may be modified or appropriately modified and combined without departing from the spirit, scope and scope of the invention.
  • the techniques of the present invention are implemented and applied.
  • Chinese cobra venom collection After the glass beaker is cleaned, it is sealed with a film and disinfected by irradiation. The Chinese spectacles tooth puncture film is forced to top on the beaker wall, and the venous gland is gently squeezed, and the venom will flow into the cup along the glass wall.
  • Chinese cobra lyx freeze powder can also be purchased from Rainbow Snake Farm in Yingtan City, Jiangxi province.
  • a suitable concentration of Chinese cobra venom was prepared in 0.5 M PBS (pH 7.8), added to a 500 ml glass flask, and sealed with a lid. The flask was placed in a water bath at 100 ° C. After boiling for 5 minutes, the heating was stopped, and ice cubes were added to the water bath to gradually cool the temperature to 20. C, the cooling rate is controlled at 5 per minute. C. The heat-treated snake venom was examined by SDS-PAGE to determine the modification result. An increase in the protein content of the molecular weight component below 10 kD should be detected.
  • Thailand Mirror venom collection After the glass beaker is cleaned, it is sealed with a film and disinfected by irradiation. Put the Thai spectacles tooth piercing film on the beaker wall, gently squeeze the venous gland, and the venom will flow into the cup along the glass wall.
  • Thai cobra lyophilized powder can also be purchased from Rainbow Snake Farm in Yingtan City, Jiangxi Republic.
  • a suitable concentration of Thai scorpion venom was prepared in 0.5 M PBS (pH 7.8), added to a 500 ml glass flask, and sealed with a lid. The flask was placed in a water bath at 100 ° C. After boiling for 5 minutes, the heating was stopped, and ice cubes were added to the water bath to gradually cool the temperature to 20. C, the cooling rate is controlled at 5 per minute. C. The heat-treated snake venom was detected by SDS-PAGE. Molecular weight groups below 10kD should be detected The protein content of the fraction is increased.
  • Example 3 Pharmacological action and mechanism of modified cobra venom
  • modified cobra venom The pharmacological effects and mechanisms of modified cobra venom were studied with modified Naja naja atra as an example.
  • the heat treatment of the venom venom After the heat treatment of the venom venom, it was formulated into a concentration of 1 mg/ml and mixed into a Western blot loading buffer.
  • the snake venom protein fraction was separated by 15% SDS-PAGE gel, stained with Coomassie blue, and photographed.
  • the protein component changes after coke venom heat treatment.
  • the results show that the heat treatment has no effect on the protein component with molecular weight above 20kD, but the protein component with molecular weight of about 18 kD is reduced (shown by solid arrow); Protein components with molecular weights below 10 kD increase (shown by open arrows).
  • the molecular weight of the target neuron toxin is about 7 kD.
  • Neurotoxin is the main analgesic and toxic component of cobra venom and is also a major component of immune regulation.
  • FIG. 2 is a comparison of the pain threshold of physical modification and modification before 30 g/kg of the venom of the venom of the present invention. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001 compared to the pre-dose pain threshold. Mirror venom
  • the pain threshold after physical administration was compared with the pre-dose pain value. A significant analgesic effect was produced 3 hours after administration, and the analgesic effect continued until 8 hours after administration.
  • Mirror venom (30 g/kg) The pain threshold after administration in the untreated group was compared with the pre-dose pain threshold, which was effective 3 h after administration, and the analgesic intensity and duration were higher than those after heat treatment.
  • Modified cobra venom It can be seen that the analgesic effect of the physically modified eye venom is stronger than that of the untreated one and the duration is prolonged.
  • the pain threshold of the blank group can rule out the error caused by the effect of prolonging the pain threshold over time.
  • Figure 3 shows the comparison of the pain threshold of physical modification and modification before cobra venom lOO g/kg.
  • the results were compared with the pre-dose pain threshold *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ie, the pain threshold and administration of the target venom snake venom (100 g/kg) in the physically modified group.
  • a significant analgesic effect was observed 3 h after administration, and the analgesic effect continued until 11 h after administration.
  • Mirror venom (100 g/kg) The pain threshold after administration in the untreated group was effective 4 h after administration compared with the pre-dose pain threshold, and the analgesic intensity and duration were worse than those of the heat treatment.
  • the analgesic effect of the physically modified venom venom has a longer duration than that of the untreated one, and the analgesic effect is significantly enhanced.
  • the pain threshold of the blank group can rule out the error caused by the effect of prolonging the pain threshold over time.
  • the delayed type hypersensitivity reaction is sensitized with 2,4-dinitrochlorobenzene (DNCB) skin, and after sensitization After 7 days, DNCB attacked the right ear of the mice again to induce an allergic reaction. After 24 hours, the neck was removed and the ears were cut. The ears were cut with the puncher at the same part of both ears, and weighed to the left and right ears. The difference in sheet weight is used as the degree of swelling.
  • DNCB 2,4-dinitrochlorobenzene
  • the target venom snake venom (300 g/kg) has a certain retardation effect on the delayed-type hypersensitivity reaction in mice after physical modification.
  • the occurrence of delayed type hypersensitivity is a release process of an inflammatory factor. It can be seen that the physically modified cobra venom has certain anti-inflammatory and anti-immune effects.
  • Table 1 The results are shown in Table 1.
  • mice The thymus and spleen of the experimental group were weighed to the spleen weight (mg) and thymus weight per 10 g of mice.
  • the present invention uses the methods of injection administration and external use to examine the anti-inflammatory and analgesic effects of the modified cobra venom, and the results also show that the venom effect of the modified snake venom is remarkably enhanced.
  • the cobra venom of Thailand also showed the effect of increasing efficiency and reducing toxicity.
  • Table 2 Effect of Chinese cobra venom on gastric spleen coefficient after intragastric administration
  • Naja naj a atra 30 ( ⁇ g/kg(+) 25.2644 ⁇ 10 ⁇ 1690 54.4699 ⁇ 4.7004
  • the modified protein of Naja naja atra obtained by the present invention showed a change in the content of protein components.
  • the modified Chinese cobra venom (Naja naja atra, lO-lOOO g/kg) obtained by the invention is physically modified, and the analgesic effect of the oral administration on the mice is obviously enhanced, and the drug is compared with the untreated group. More effective.
  • the modified Chinese cobra venom (Naja naja atra, 30-1000 g/kg) obtained by the invention is physically modified, and the intragastric administration has a delayed effect on the delayed type hypersensitivity reaction in the mouse, but the same dose is not The modified venom effect is not obvious.
  • Kunming mice were given gavage venom by mouth venom, divided into 6 dose groups, and the death after administration was determined.
  • the Bliss method calculates the median lethal dose (LD50).
  • LD50 median lethal dose
  • the results showed that: the untreated Chinese cobra venom was orally administered with an LD50 dose of 102 mg/kg; the heat-treated Chinese cobra venom was orally administered with LD 5 .
  • the dose was 122 mg/kg. This result suggests that the toxicity of the target venom of the venom has decreased. Therefore, the acute toxicity of the modified Chinese cobra venom (Naja naja atra) of the present invention is decreased.
  • Example 5 Modified cobra venom composition pharmaceutical dosage form and preparation
  • Modified cobra venom 30-300 ⁇ ⁇ sterilized physiological saline lml is prepared according to the conventional injection preparation method.
  • Modified cobra venom 30-50 ( ⁇ g/tablet, 50% gelatin, 5% xylitol, 1.5% flavoring agent prepared according to the conventional film preparation method.
  • the physical modification method of the modified cobra venom and the cobra venom and the use thereof in the preparation of the oral, topical and injectable drugs have been described by way of examples, and it will be obvious to those skilled in the art that the present invention can be carried out without departing from the spirit, scope and scope of the invention.
  • the physical properties of the modified cobra venom and cobra venom The modification method and the use in the preparation of oral, topical, and injectable drugs are modified or appropriately changed and combined to realize the present technology. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the spirit, scope and content of the invention.

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Description

改性眼镜蛇毒及其制备方法和用途
本申请要求于 2011年 3月 18日提交中国专利局、 申请号为
201110065634.1、 发明名称为"眼镜蛇毒物理改性方法和在制备镇痛或免疫抑 制药物中的应用 "的中国专利申请的优先权， 其全部内容通过引用结合在本申 请中。 技术领域
本发明属于药物制备技术领域，具体涉及一种改性眼镜蛇毒及眼镜蛇毒的 物理改性方法和在制备口服、 外用和注射剂药物中的用途。 背景技术
蛇毒是由蛇的毒腺分泌的多种组分构成的复杂混合物。 蛇毒的干物质中 90%以上是蛋白质， 是其毒性和其生物学活性的主要成分。 祖国传统医学认为 目艮镜蛇及其毒性成分可通经络， 祛风湿， 并具有强身健体之功效， 早在二十世 纪初， 人们就开始应用蛇毒来緩解恶性肿瘤疼痛、 神经痛和关节痛， 随后出现 一系列关于蛇毒有类似吗啡样镇痛作用的报道。 随着研究的不断深入， 现在， 蛇毒及其制品已应用于临床， 除了镇痛作用外， 在抗炎、 抗风湿、 抗肿瘤以及 在神经系统疾病治疗方面都有 4艮好的疗效。 中华眼镜蛇毒 ( Naja naja atra )和 泰国目艮镜蛇毒（Naja Kaouthia )两种眼镜蛇毒所含成分接近， 药理作用和毒性 十分相似， 两者可以开发成同类型的药物和具有相同的临床用途。 发明内容
本发明目的在于提供一种改性眼镜蛇毒， 提供了一种新型的镇痛药物。 为了解决现有技术中的这些问题， 本发明提供的技术方案是：
一种改性眼镜蛇毒，其特征在于所述眼镜蛇毒来自中华眼镜蛇毒或泰国眼 镜蛇毒，所述改性眼镜蛇毒是将眼镜蛇毒 60-100 °C加热 5-120分钟后冷却后得 到， 且所述改性眼镜蛇毒用 SDS-PAGE检测时出现特征性的小分子量蛋白的 增加。 经试验证实该改性眼镜蛇毒药效增强而毒性下降。
本发明的另一目的在于提供一种含有改性眼镜蛇毒的组合物，其特征在于 所述组合物以其组分重量百分比计含有：
改性眼镜蛇毒 0.01 ~ 10重量分数；
药学上可接受的载体 90 ~ 99.99重量分数。
优选的， 所述组合物的剂型选自口服剂型或外用剂型或注射剂。
优选的， 所述口服剂型选自口服液、 胶嚢剂和口服药膜； 所述外用剂型选 自涂剂、 巴布剂、 喷雾剂， 所述注射剂型选自粉针剂和水针剂。
本发明的又一目的在于提供一种改性眼镜蛇毒的改性方法，其特征在于所 述改性方法为物理改性方法， 包括将眼镜蛇毒在 60-100°C加热 5-120分钟后进 行冷却的步骤。
优选的， 所述改性方法中眼镜蛇毒溶于 0.5 M PBS緩沖液， 调整溶液的 PH值在 7.8左右进行。
本发明的又一目的在于提供一种改性眼镜蛇毒在制备镇痛和免疫抑制药 物方面的应用。
本发明涉及了一种目艮镜蛇毒的物理改性方法和在制备口服、注射和外用药 物中的用途。本发明的眼镜蛇毒液经过热处理及其它辅助条件改变部分组分含 量， 达到增效减毒目的； 所述改性蛇毒药物包括口服液、 胶嚢、 药膜、 涂剂、 巴布剂、 喷雾剂和注射剂等各种剂型。
本发明眼镜蛇毒来自于中华眼镜蛇毒 ( Naja naja atra )和泰国目艮镜蛇毒
( Naja Kaouthia ), 通过以下方法可以得到眼镜蛇毒： 从人工饲养的中华或泰 国目艮镜蛇采集毒液， 或从市场购买眼镜蛇毒冻干粉。 目艮镜蛇毒包括中华眼镜蛇 和泰国眼镜蛇所产蛇毒。
本发明的所述药物还包括各种单方和复方制剂。尽管中华眼镜蛇蛇毒在临 床上已用于治疗风湿和緩解恶性肿瘤疼痛、神经痛和关节痛等，但目前还没有 文献报道中华目艮镜蛇总毒（ Naja naja atra )和泰国目艮镜蛇总毒（ Naja Kaouthia ) 在经过热处理后可以减毒增效。也没有报道这种改性的蛇毒可以通过口服、鼻 (口） 粘膜给药、 皮肤给药和注射给药途径进行临床用药而发挥疗效。
目艮镜蛇毒的物理改性方法： 将眼镜蛇毒溶液于 0.5 M PBS ( pH7.8 ) 中， 60-100°C加热 120分钟左右 （范围 5-120分钟）， 将加热的眼镜蛇毒液快速置 于冰中冷却（或逐步冷却）。 经过这种热处理后的蛇毒用 SDS-PAGE检测时出 现特征性的小分子量蛋白的增加。
本发明研究发现经过物理改性后中华眼镜蛇毒的镇痛作用明显增强，并可 以延迟迟发型超敏反应。 蛇毒改性的另一个作用是毒性有所下降。 这一发现， 将对蛇毒的镇痛作用，免疫调节作用的研究和药物开发产生重大的影响， 并有 临床实用价值， 为中华眼镜蛇毒开辟临床新的用途。
相对于现有技术中的方案， 本发明的优点是：
本发明中眼镜蛇毒（包括中华泰国目艮镜蛇毒和泰国目艮镜蛇毒）经过物理改 性后， 不仅镇痛作用更有效而持久， 还具有较好的抗炎和免疫调节作用。 本发 明目艮镜蛇毒（包括中华泰国目艮镜蛇毒和泰国眼镜蛇毒）经过物理改性后用量少， 有效范围大， 从 lO-lOOO g/kg都有很好的镇痛和免疫调节作用作用， 安全范 围扩大。 本发明眼镜蛇毒（包括中华泰国目艮镜蛇毒和泰国目艮镜蛇毒）经过物理 改性后多种给药途径包括皮肤给药、 口服、 口 （鼻）粘和注射等途径给药均有 效。
综上所述， 本发明公开了一种目艮镜蛇毒 (Naja naja atra)物理改性的方法， 经过物理改性的目艮镜蛇毒， 在镇痛， 抗炎抑制免疫方面的药效增强， 作用更持 久。 目艮镜蛇毒取自人工繁殖饲养的中华或泰国目艮镜蛇的毒液。本发明的研究发 现经物理改性后的目艮镜蛇毒（ lO-lOOO g/kg ) 范围内镇痛、 抗炎抑制免疫作用 明显， 具有用量小、 安全、 可口服、 可注射、 可透过口鼻粘膜和皮肤给药等优 点， 具有较大的临床实用价值。 附图说明
图 1为本发明实施例眼镜蛇毒加热处理后蛋白组分的变化；
图 2为本发明实施例目艮镜蛇毒 30 g/kg经物理改性与改性前痛阈值比较 图； 其中， *P<0.05, **P<0.01, ***P<0.001与给药前痛阈值相比。
图 3为本发明实施例目艮镜蛇毒 lOO g/kg经物理改性与改性前痛阈值比较 图。 具体实施方式
本发明公开了一种改性目艮镜蛇毒及眼镜蛇毒的物理改性方法和在制备口 月良、 外用和注射剂药物中的用途， 本领域技术人员可以借鉴本文内容， 适当改 进工艺参数实现。特别需要指出的是， 所有类似的替换和改动对本领域技术人 员来说是显而易见的， 它们都被视为包括在本发明。 本发明的产品、 方法及应 用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精 神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应 用本发明技术。
为了使本领域的技术人员更好地理解本发明的技术方案，下面结合具体实 施例对本发明作进一步的详细说明。 实施例 1 中华眼镜蛇毒的改性处理
中华眼镜蛇毒采集： 玻璃烧杯清洁后用薄膜封口， 辐照消毒。 将中华眼镜 蛇牙齿刺破薄膜加力顶在烧杯壁上， 轻轻挤压毒腺部位，毒液将沿玻壁流入杯 中。 中华眼镜蛇毒冻干粉也可从江西鹰潭市彩虹蛇场购得。
用 0.5MPBS ( pH7.8 )配制合适浓度的中华眼镜蛇毒液， 加入到 500ml玻 璃烧瓶中， 加盖密封。 置烧瓶于 100 °C水浴锅中， 煮沸 5分钟后， 停止加热， 在水浴锅中加入冰块逐步使温度冷却至 20。C, 降温速度控制在每分钟 5。C。 加热处理的蛇毒用 SDS-PAGE检测改性结果。应该检测到 10kD以下分子量组 分的蛋白含量增加。 实施例 2泰国眼镜蛇毒的改性处理
泰国目艮镜蛇毒采集： 玻璃烧杯清洁后用薄膜封口， 辐照消毒。 将泰国眼镜 蛇牙齿刺破薄膜加力顶在烧杯壁上， 轻轻挤压毒腺部位，毒液将沿玻壁流入杯 中。 泰国眼镜蛇毒冻干粉也可从江西鹰潭市彩虹蛇场购得。
用 0.5MPBS ( pH7.8 )配制合适浓度的泰国目艮镜蛇毒液， 加入到 500ml玻 璃烧瓶中， 加盖密封。 置烧瓶于 100 °C水浴锅中， 煮沸 5分钟后， 停止加热， 在水浴锅中加入冰块逐步使温度冷却至 20。C, 降温速度控制在每分钟 5。C。 加热处理的蛇毒用 SDS-PAGE检测改性结果。应该检测到 10kD以下分子量组 分的蛋白含量增加。 实施例 3 改性眼镜蛇毒的药理作用和机理考察
以下以改性中华眼镜蛇毒 ( Naja naja atra ) 为例进行改性眼镜蛇毒的药理 作用和机理考察。
目艮镜蛇毒加热处理后， 配制成 1 mg/ml浓度， 混入 Western blot加样緩沖 液。 蛇毒蛋白组分用 15%SDS-PAGE胶分离， 考马氏亮蓝染色， 摄片。 如图 1 所示为眼镜蛇毒加热处理后蛋白组分的变化， 结果显示加热处理对分子量 20kD以上的蛋白组分没有影响，但是分子量为 18 kD左右的蛋白组分减少（实 心箭头所示 ); 分子量 10 kD以下的蛋白组分增加（空心箭头所示）。 目艮镜蛇短 链神经毒素的分子量为 7 kD左右。 神经毒素是眼镜蛇毒中主要的镇痛和毒性 成分， 也是免疫调节的主要成分。
如图 2所示为本发明实施例目艮镜蛇毒 30 g/kg经物理改性与改性前痛阈值 比较。 与给药前痛阈值相比 *P<0.05, **P<0.01, ***P<0.001。 即目艮镜蛇毒
( 30 g/kg )物理改性组给药后痛阈值与给药前痛阁值相比较。 在给药后 3h产 生明显的镇痛作用， 且镇痛作用持续到给药后 8h。 目艮镜蛇毒（ 30 g/kg ) 未处 理组给药后痛阈值与给药前痛阈值相比， 在给药后 3h起效， 镇痛作用强度和 持续时间都比经加热处理后形成的改性眼镜蛇毒差。可见， 经过物理改性的眼 镜蛇毒的镇痛作用较未处理的药效增强、持续时间延长。 空白组的痛阈值可以 排除随时间延长对痛阈值的影响所产生的误差。
如图 3所示为眼镜蛇毒 lOO g/kg经物理改性与改性前痛阈值比较图。 结 果与给药前痛阈值相比 *P<0.05, **P<0.01, ***P<0.001 ,即目艮镜蛇毒（ lOO g/kg ) 物理改性组给药后痛阈值与给药前痛阈值相比， 在给药后 3h产生明显的镇痛 作用， 且镇痛作用持续到给药后 llh。 目艮镜蛇毒（ lOO g/kg ) 未处理组给药后 痛阈值与给药前痛阈值相比， 在给药后 4h起效， 镇痛作用强度和持续时间都 比加热处理的差。可见， 经过物理改性的目艮镜蛇毒的镇痛作用较未处理的药效 持续时间增长，且镇痛作用明显增强。 空白组的痛阈值可以排除随时间延长对 痛阈值的影响所产生的误差。
迟发型超敏反应是采用 2,4-二硝基氯苯（DNCB )皮肤致敏， 与致敏后第 7天以 DNCB再次攻击小鼠右耳诱发过敏反应， 24h后， 脱颈推处死， 分别剪 下双耳， 用打孔器在两耳相同部位打下圓耳片， 称重， 以左、 右耳片重量差值 作为肿胀度。
结果提示， 与生理盐水对照组相比， 目艮镜蛇毒（ 300 g/kg )经物理改性后 对小鼠迟发型超敏反应具有一定的延迟作用。迟发型超敏反应的发生是一个炎 症因子的释放过程，可见，经物理改性的眼镜蛇毒具有一定的抗炎抗免疫作用。 结果见表 1。
表 1中华眼镜蛇毒灌胃给药后对小鼠迟发型超敏反应的影响
组别 剂量 耳缘肿胀度
Saline 一 0.007620士 0.003862
Naja naja atra 300μ8¾ (+) 0.004267士 0.000841*
Naja naja atra 30(^g/kg 0.005511±0.002387
Naja naja atra 100μ8¾ (+) 0.004980士 0.001933
Naja naja atra 3(^g/kg(+) 0.004625±0.002994
+, 加热处理； *P<0.05 , 与生理盐水对照组相比
对实验组小鼠的胸腺， 脾脏称重， 以每 10g小鼠的脾重（mg )和胸腺重
( mg )作为脾指数和胸腺指数。 结果表明， 目艮镜蛇毒经物理改性组， 与未处 理组对 d、鼠的脏脾系数没有显著的影响。 结果见表 2。
另夕卜， 本发明采用注射给药和外用的方法， 考察改性眼镜蛇毒的抗炎和镇 痛作用， 结果也表明改性后的蛇毒药效显著增强。 经同样的处理， 泰国眼镜蛇 毒也出现增效减毒的效果。 表 2中华眼镜蛇毒灌胃给药后对小鼠脏脾系数的影响
组别 剂量 胸腺指数 脾脏指数
Saline 一 26.5252士 9.3858 50.2513士 12.8458
Naja naj a atra 30(^g/kg(+) 25.2644士 10· 1690 54.4699士 4.7004
Naja naj a atra 30(^g/kg 25.7321士 7.2370 43.5058士 6.6686
Naja naj a atra 10(^g/kg(+) 26.7109士 5.0391 53.8832±13.0970
Naja naj a atra 3(^g/kg(+) 30.0296士 7.8201 49.3530士 11.1497 + , 力口热处理。
从上面的实验结果证实： 本发明得到的改性后的中华眼镜蛇毒（Naja naja atra ) 改性处理后出现蛋白组分含量发生变化。 本发明得到的改性后的中华眼 镜蛇毒（ Naja naja atra, lO-lOOO g/kg )经物理改性后， 口服给药对小鼠的镇痛 作用明显增强，且与未处理组相比药效更持久。本发明得到的改性后的中华眼 镜蛇毒（ Naja naja atra, 30-1000 g/kg )经物理改性后， 灌胃给药对小鼠的迟发 型超敏反应有延迟作用，而同剂量未经改性处理的蛇毒作用不明显。 实施例 4 改性眼镜蛇毒的药物毒性考察
昆明种小鼠灌胃给予目艮镜蛇毒，分 6个剂量组，测定给药后死亡情况， 按
Bliss法计算半数致死量（LD50 )。 结果显示： 未处理的中华眼镜蛇毒口服的 LD50剂量为 102mg/kg; 加热处理过的中华眼镜蛇毒口服的 LD5。剂量为 122mg/kg。 该结果提示物理改性后的目艮镜蛇毒毒性有所下降。 所以， 本发明改 性处理后的中华眼镜蛇毒（Naja naja atra ) 急性毒性下降。 实施例 5 改性眼镜蛇毒组合物药物剂型及制备
( 1 )外用涂剂：
改性眼镜蛇毒 10-lOOmg/lOO ml, 50%丙二醇， 10%乙醇， 1%增稠剂， 0.26% 苯扎氯铵按照常规的涂剂制备方法制备而成。
( 2 )注射剂： 改性眼镜蛇毒 30-300μ§, 灭菌生理盐水 lml按照常规的注 射剂配制方法制备而成。
( 3 ) 口腔喷剂： 改性目艮镜蛇毒 10-100mg/ 100 ml, 0.1%磷酸二氢钠， 5% 木糖醇， 1.5%矫味剂按照常规的喷剂配制方法制备而成。
( 4 ) 口服药膜： 改性眼镜蛇毒 30-50(^g/片， 50%明胶， 5%木糖醇， 1.5% 矫味剂按照常规的药膜配制方法制备而成。 本发明提出的改性眼镜蛇毒及眼镜蛇毒的物理改性方法和在制备口服、外 用和注射剂药物中的用途已通过实施例进行了描述，相关技术人员明显能在不 脱离本发明内容、精神和范围内对本文所述的改性眼镜蛇毒及眼镜蛇毒的物理 改性方法和在制备口服、外用和注射剂药物中的用途进行改动或适当变更与组 合， 来实现本发明技术。 特别需要指出的是， 所有相类似的替换和改动对本领 域技术人员来说是显而易见的， 它们都被视为包括在本发明的精神、 范围和内 容中。

Claims

权 利 要 求
1. 一种改性眼镜蛇毒， 其特征在于， 所述改性目艮镜蛇毒来源于中华眼镜 蛇毒或泰国眼镜蛇毒，所述改性眼镜蛇毒是将眼镜蛇毒 60-100 °C加热 5-120 钟后冷却后得到， 且所述改性眼镜蛇毒用 SDS-PAGE检测时出现特征性的小 分子量蛋白的增加。
2. 一种含有改性眼镜蛇毒的组合物， 其特征在于所述组合物以其组分重 量百分比计含有：
改性眼镜蛇毒 0.01 ~ 10重量分数；
药学上可接受的载体 90 ~ 99.99重量分数。
3. 根据权利要求 2所述的含有改性眼镜蛇毒的组合物， 其特征在于所述 组合物的剂型选自口服剂型或外用剂型或注射剂。
4. 根据权利要求 2所述的含有改性眼镜蛇毒的组合物， 其特征在于所述 口服剂型选自口服液、 胶嚢剂和口服药膜； 所述外用剂型选自涂剂、 巴布剂、 喷雾剂， 所述注射剂型选自供肌肉或皮下注射给药使用的粉针剂和水针剂。
5. 一种改性眼镜蛇毒的改性方法， 其特征在于所述改性方法为物理改性 方法， 包括将眼镜蛇毒在 60- 100 °C加热 5- 120分钟后进行冷却的步骤。
6. 根据权利要求 5的改性方法， 其特征在于所述改性方法中眼镜蛇毒溶 于 0.5 M PBS緩沖液， 调整溶液的 PH值在 7.8左右进行。
7. 一种权利要求 1所述的改性眼镜蛇毒在制备镇痛药物或免疫抑制药物 方面的应用。
-I-
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