WO2005004874A1 - Stable tetrodotoxin freeze drying medicinal preparation - Google Patents

Stable tetrodotoxin freeze drying medicinal preparation Download PDF

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Publication number
WO2005004874A1
WO2005004874A1 PCT/CN2004/000736 CN2004000736W WO2005004874A1 WO 2005004874 A1 WO2005004874 A1 WO 2005004874A1 CN 2004000736 W CN2004000736 W CN 2004000736W WO 2005004874 A1 WO2005004874 A1 WO 2005004874A1
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Prior art keywords
tetrodotoxin
preparation
content
preparation according
solution
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English (en)
French (fr)
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Xiao Zhang
Yuhong Kang
Xiaoyan Huang
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Nanning Maple Leaf Pharmaceutical Co Ltd
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Nanning Maple Leaf Pharmaceutical Co Ltd
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Priority to JP2006519747A priority Critical patent/JP2007507422A/ja
Priority to CNB200480016097XA priority patent/CN100457110C/zh
Priority to EP04738334A priority patent/EP1645277A4/en
Priority to MXPA06000243A priority patent/MXPA06000243A/es
Priority to BRPI0412002-7A priority patent/BRPI0412002A/pt
Priority to CA2529598A priority patent/CA2529598C/en
Publication of WO2005004874A1 publication Critical patent/WO2005004874A1/zh
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/529—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim forming part of bridged ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • 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
    • 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
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
    • A61P25/36—Opioid-abuse
    • 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
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a lyophilized preparation for medical use, and more particularly to a stable lyophilized preparation containing tetrodotoxin for human injection.
  • BACKGROUND Tetrodotoxin is one of the non-protein neurotoxins found in nature. It can bind to the sodium ion channel SS1 / SS2 of nerve cell membrane with high selectivity and high affinity. It has been in the research fields of neuropharmacology and muscle physiology for decades. It is widely used as a pharmacological tool medicine, and the typical product on the market is 1 mg tetrodotoxin-containing citrate solid lyophilized powder (the company's article number: T5881) provided by Sigma-Aldrich.
  • tetrodotoxin injection aqueous solution
  • tetrodotoxin injection aqueous solution
  • tetrodotoxin injection is sensitive to temperature and is susceptible to degradation by temperature. The higher the temperature, the faster the degradation.
  • the content of the effective active substance tetrodotoxin is less than 90% of its stated amount or the relative content of the relevant substance exceeds the limit prescribed by the medical standard (greater than the main peak area of the control solution), it does not meet the clinical use requirements.
  • the tetrodotoxin injection is below the prescribed limit after being left for 3 days under the 4CTC condition, which does not meet the medical requirements.
  • the content of tetrodotoxin injection decreased from 100% in January to 95.34% and decreased by 4.66% after being left for 1 month.
  • the content of the related substances was larger than the area of the main peak of the control solution and exceeded the specified limit.
  • the area of the main peak of the control solution is based on the content of 0 days as i 00 %
  • Formula citrate mannitol Formula citrate mannitol White sparse muffins, white sparse muffins at 40'C, white sparse muffins at 40'C, but slow white sparse muffins, room temperature Under the appearance of 1 day, it was almost completely contracted and changed. After S 10 days, no obvious change in appearance was observed. Slow shrinkage changed to form crystals. No obvious change was observed in storage. The appearance and appearance did not meet the requirements. The appearance is in compliance with regulations. On the bottle wall, the appearance is not acceptable. Compliance.
  • Micro related substances with fl related substances Micro with fl related substances with fi related substances with fl (%)
  • Stability additive We gradually realized that we must start with the chemical structure of tetrodotoxin and open up a new way to improve the temperature stability of the practical formulation of tetrodotoxin.
  • tetrodotoxin 10 hydrogen-12- (diameter methyl) -2-imino-5, 9: 7, 10 ⁇ -dimethyl bridge-10 ⁇ ⁇ - (1,3) dibridged oxo (6 , 5-d) -pyrimidine-4,7,10,11,12-pentanol, the molecular formula is CuH 17 N 3 0 8 , and the molecular weight is 319.28.
  • the structural formula is as follows:
  • Tetrodotoxin's structural formula Tetratoxin turns black above 220 ° C, but does not decompose, [a] ⁇ 5 -8.64 (C 8.55 in dilute acetic acid).
  • Tetrodotoxin is quite stable to heat in the solid state, but its aqueous solution, especially a low-concentration dilute acid solution, is stable. Poor qualitative (Kang, et al.
  • the object of the present invention is to provide a stable freeze-dried preparation of tetrodotoxin or its analog.
  • the present invention provides a stable medicinal freeze-dried preparation, which contains tetrodotoxin or its analogue as a biologically active substance, and the content of said tetrodotoxin or its analogue is 0.5-60 ⁇ ⁇ /
  • the formulation further contains a compound capable of reducing the tetrodotoxin or its analog C-4 hydroxyl activity, preferably a glycosidic bond containing any one of a disaccharide, a glycan or a derivative thereof or a mixture thereof.
  • a compound capable of reducing the tetrodotoxin or its analog C-4 hydroxyl activity preferably a glycosidic bond containing any one of a disaccharide, a glycan or a derivative thereof or a mixture thereof.
  • tetrodotoxin will be converted into 4-epi-tetrodotoxin and 4,9-anhydrotetrodotoxin in a 90 ° C aqueous solution, and partially converted to tetrodoic acid.
  • the only difference is the stereo position of the 4-hydroxy group; 4,
  • the molecular formulas of 9-anhydrotetradotoxin, tetrodotoxin and 4-epiotetrodotoxin are only one H 2 0 different, and the molecular weight is 18, but their biological activity is There are significant differences.
  • the toxicity of tetrodotoxin is 4500 rat units / mg
  • the toxicity of 4-epi-tetrodotoxin is 710 rat units / mg
  • the toxicity of 4,9-anhydrotetrodotoxin is only 92 rat units / mg (Toxicon.
  • tetrodotoxin is only about 1/700 less tetrodotoxin.
  • 4-epi-tetrodotoxin, and 4,9-anhydrotetrodotoxin once tetrodotoxin is converted to 4-epi-tetrodotoxin and 4,9-anhydrotetrodotoxin, the original biological The activity will greatly decrease and lose its medicinal value.
  • Question 3 What is the chemical mechanism for the conversion of tetrodotoxin to 4-epi-tetrodotoxin and 4,9-anhydrotetradotoxin?
  • C-4 The specificity of C-4 is easy to see from the molecular structure of tetrodotoxin.
  • C-4 which has an OH bond and an upright bond hydrogen atom, is ortho to the nitrogen. Therefore, the hydroxyl group on C-4 has a special chemical And physiological activity.
  • H + When H + is present in the solution, H + will combine with the oxygen atom on the C-4 hydroxyl group, and change from structure A to structure B with a positive charge, and structure B loses a molecule of water to generate a structure with positive charge at C-4. .
  • the structure C is combined with water molecules, and the water molecules can either attack from the original position where the water molecules were removed to derive the structure E, or attack from the opposite side of the position where the water molecules are removed to derive the structure D.
  • Removal of H + from structure E produces the original tetrodotoxin, structure A, and removal of H + from structure D produces structure F.
  • the difference between structure F and structure A is that the positions of the hydrogen atom and the hydroxyl group are interchanged.
  • the H atom of C-4 is an upright bond and the hydroxyl group is a flat bond.
  • structure F the The H atom is a flat bond and the hydroxyl group is an upright bond.
  • Both structures A and F are chemically referred to as epimers.
  • the mechanism of tetrodotoxin isomerization is as follows:
  • tetrodotoxin The mechanism of isomerization of the 4-hydroxy group of tetrodotoxin A is called “tetrodotoxin” and is the main component of tetrodotoxin (TTX) obtained from puffer fish in nature. Structure F is commonly referred to as 4-epi-tetrodotoxin. Because the hydroxyl group on C-4 is very close to the hydroxyl group on C-9, it acts on H +. It is easy to lose a molecule of water to produce more stable 4,9-anhydrotetrodotoxin. The three so-called “tetrodotoxins” have only slightly different chemical properties, but their biological activities are significantly different.
  • 4-Deoxytetrodotoxin is very stable. HPLC analysis shows that its aqueous solution does not change after boiling for 2 hours, but its 11) 5 () is 3336.5 g kg, which is 330 times less toxic than tetrodotoxin. Furthermore, the analgesic effect of the mouse acetic acid twist method was measured, and the results showed that the analgesic effect was about 330 times smaller than that of tetrodotoxin.
  • the inventor has explored a method for solving the above problems. Because the stereo position of the C-4 hydroxyl group is one of the key groups for the biological activity of tetrodotoxin, the inventor of this application uses this as a starting point to find a property that can be pharmaceutically acceptable without changing the C-4 hydroxyl group. Substances with stereo positions, thereby preventing their epimerization from occurring. First consider that the hydrogen atom on the hydroxyl group can form a hydrogen bond with a highly electronegative atom such as an oxygen atom. There are 6 electrons in the outer layer of the oxygen atom.
  • Tetrodotoxin lactose Based on the same principle, glycans such as condensed glucose, dextran, etc. and their derivatives such as hydroxyethyl starch, hydroxypropyl cyclodextrin, etc. also contain furoside bonds, and the molecular structure is similar to that of tetrodotoxin. It should also have a stabilizing effect on tetrodotoxin. Our research found that glycans do have this effect.
  • the disaccharide of the monosaccharide ⁇ anomer epoxy atom and the dipole of the hydroxyl group on C-1 are parallel to each other.
  • the technical solution of the present invention is as follows ⁇
  • the present inventor has invented a pharmaceutical formula and a preparation method for providing a biologically active ingredient, tetrodotoxin, which is not stable enough in an aqueous solution or a citrate lyophilized form in the form of a lyophilized preparation, thereby To achieve the purpose of long-term storage at room temperature. In clinical application, it can be regenerated by adding an aqueous carrier acceptable to the human body for injection administration.
  • the stable medicinal tetrodotoxin freeze-dried preparation according to the present invention is a pharmaceutical composition containing a trace biologically active substance tetrodotoxin (Ttrodotoxin TTX), a stabilizer, and a co-solvent.
  • the tetrodotoxin is in a lyophilized preparation composition The amount is only 0.5-60 ⁇ ⁇ per dose, and the rest are stabilizers and co-solvents.
  • the tetrodotoxin mentioned above includes tetrodotoxin and its analogues, tetrodotoxin, amine tetrodotoxin, methoxytetrodotoxin, ethoxytetrodotoxin, tetrodotoxin with biologically active ingredients is obtained from the ovaries and liver of puffer fish in marine animals. It can be obtained from other species, or from other species, such as amphibians, flat animals, worms, neoformans, echinoderms, starfishes, hairy jaws, arrowworms, software animals, gastropods, and some bacteria. Extracted from bacteria such as Vibrio alginolyticus.
  • the extraction method can refer to the method of the existing public literature, for example, the patent of the applicant (Zhou, et al., US 6,552,191). Its analogs are compounds obtained by structural modification with tetrodotoxin.
  • the tetrodotoxin which is a bioactive substance that meets the requirements for safe injection in the human body, has a very small content, each dose is only 0.5 ⁇ ⁇ to 60 ⁇ ⁇ , which cannot be made into a lyophilized preparation, and usually must be added to a pharmaceutically acceptable Excipients to increase the concentration of the solution before lyophilization.
  • the substance capable of preventing epimerization of tetrodotoxin and having a stabilizing effect is a disaccharide such as lactose, sucrose, maltose, and cellobiose, or a glycan such as dextran, or a derivative thereof such as hydroxyethyl starch , Hydroxypropylcyclodextrin, etc., can be used alone or in combination of two or more, the dosage of each dose is 5mg ⁇ 100mg.
  • the biologically active substance tetrodotoxin is almost insoluble in water.
  • a co-solvent that can increase the solubility of tetrodotoxin must be added. From the analysis of the chemical structure of tetrodotoxin, it is an organic base, so it is soluble in acids, but strong acids will break it down. After research, only non-volatile organic acids such as citric acid, malic acid, tartaric acid, lactobionic acid, etc. Ideally, they can form organic acid salts with tetrodotoxin and dissolve in water, and easily control their acidity.
  • 0005mg ⁇ Test proves that the amount of co-solvent used in the present invention should make the pH of the solution before lyophilization is 3. 0 ⁇ 6. 0, usually the dosage per dosage is 0. 00005 ⁇ 0. 0005mg.
  • the present invention is obtained by freeze-drying a solution containing a biologically active substance, tetrodotoxin, and an aqueous solution or a water-soluble solvent of a disaccharide or a polysaccharide or a derivative thereof, to obtain a solid (Reminton's Pharmaceutical Sciences. Seventeenth Edition. 1985 1314.)
  • the aqueous solution described above is easy to prepare. After passing through a 0.22 ⁇ membrane, and then passing through an ultrafiltration filter with a molecular weight of 10,000, the lyophilized preparation can be sterile and pyrogen-free after freeze-drying.
  • the resulting lyophilized preparation is stable and can prevent the conversion of the biologically active substance tetrodotoxin for a long time.
  • a calculated amount of co-solvent is usually added to control the pH value between 3. 0 ⁇ 6. 0 If pH is below 3.0, can be adjusted with dilute sodium hydroxide (potassium) solution or sodium (potassium) salt of the corresponding co-solvent. The amount should be determined according to the pH value of the solution before freeze-drying. If the pH value of the solution is already between 3. 0 ⁇ 6. 0, it is not necessary to adjust the pH value.
  • the method for preparing a freeze-dried preparation containing trace tetrodotoxin according to the present invention is: dissolving a co-solvent, a stabilizer, and a pH adjuster in water, dissolving a trace tetrodotoxin in a metered co-solvent, and adding it to the stabilizer solution. Add water to the specified volume, mix well, and determine the pH value between 3. 0 ⁇ 6.0, otherwise adjust the pH value with sodium hydroxide (potassium) or the corresponding organic sodium (potassium) salt. After ultrafiltration, it is quantitatively filled into glass vials, which can be obtained by semi-stopping, freeze-drying, pressing, and capping.
  • the freeze-dried preparation composition described above is regenerated with a human-acceptable aqueous solution carrier to obtain a sterile, pyrogen-free clear liquid that can be used for intramuscular or subcutaneous injection administration.
  • the aqueous solution carrier is sterilized water for injection or other aqueous carrier.
  • the amount of the carrier is 0.5-5 ml, and the optimal amount is 1 to 2 ml.
  • the specific preparation process is-
  • Sterile, pyrogen-free clear solutions are obtained after sterilizing filtration (such as the filtration system of Millipore) and ultrafiltration (such as the ultrafiltration system of Pull).
  • Adding stabilizer disaccharide or glycan or its derivative to the formulation can effectively protect the tetrodotoxin from epimerization during storage, and can effectively prevent the conversion of tetrodotoxin to 4-epitoxin and 4,9 -Dehydrated tetrodotoxin, product quality is guaranteed.
  • the product contains very little moisture, which improves the stability of the product.
  • Example 1 A freeze-dried preparation of tetrodotoxin and disaccharides (lactose, sucrose, maltose, cellobiose) was prepared in the amounts shown in Table 7.
  • Formula 1 uses fructose (monosaccharide) as an excipient, and formulas 2, 3, 4, and 5 use disaccharide as a stabilizer (also serves as an excipient).
  • Dissolve citric acid (co-solvent) and stabilizer in water for injection dissolve tetrodotoxin in citric acid solution, and then add the stabilizer solution to it, add water for injection to the specified volume, stir well, and adjust the pH to 4.0 , Sterilizing filtration, ultrafiltration, quantitative filling in glass vials, half stoppered, sent to the freeze dryer, pre-freeze until the temperature of the article reaches below -40 ⁇ , open the cold trap to cool, so that the cold trap temperature drops to -50 ° Below C, start the vacuum pump, maintain the vacuum level below 5 Pa, and naturally increase the temperature to the set value for at least 24 hours, and then naturally increase the temperature to 30 ° C for at least 10 hours. It can be obtained by pressing and capping. Table 7
  • the tetrodotoxin lyophilized preparation and tetrodotoxin injection (water injection) obtained above were simultaneously tested for stability at 4 ° C, and samples were taken at 1, 3, 5, and 10 days, and the tetrodotoxin content was determined by HPLC. The substance content is compared with the measurement result on day 0, and the results are shown in Table 8. Test results of tetrodotoxin freeze-dried agent and tetrodotoxin injection at different temperatures
  • Formula 1 uses fructose as an excipient.
  • the appearance of the product does not meet the requirements.
  • the content of tetrodotoxin will gradually decrease from 100% (0 days) to 95.99% (10 days), a decrease of 4.01. %,
  • the content of the relevant substance exceeds the area of the main peak of the control solution after 5 days, which does not meet the requirements. Therefore, fructose cannot prevent the epimerization of tetrodotoxin and cannot solve the stability of tetrodotoxin.
  • Formulas 2, 3, 4, and 5 when using disaccharides such as lactose, sucrose, maltose, and cellobiose as stabilizers did not show significant changes in the tetrodotoxin content and related substance content after 10 days under the high temperature test conditions of 40 ° C.
  • the content of tetrodotoxin is 99.97%, 99.56%, 99.18%, and 99.75%, and the content of related substances is less than the main peak area of the control solution, which meets the requirements of medical standards and greatly improves the stability, indicating that the disaccharide can prevent the anisotropy of tetrodotoxin.
  • Formula 6 * is a water injection.
  • Formula 1 uses fructose as an excipient.
  • the appearance of the preparation is unsatisfactory.
  • the content of tetrodotoxin decreases during storage. After 12 months, the content decreases from 100% (0 months) to 83.19%, a decrease of 16.81%.
  • the related substances after 2 months The content has exceeded the main peak area of the control solution, does not meet the requirements, and cannot be used for medicine.
  • Formulations 2, 3, 4, and 5 each use disaccharides such as lactose, sucrose, maltose, and cellobiose as stabilizers. When left at room temperature for 12 months, their appearance, tetrodotoxin content, and related substance contents have not changed significantly.
  • the content of tetrodotoxin was 101.92%, 99.89%, 99.87%, and 99.47%, and the content of related substances was less than the main peak area of the control solution, which met the requirements of medical standards.
  • Formula 6 A is a water injection. Under the same conditions, the tetrodotoxin content has dropped significantly, from 12% (0 months) to 73.38%, a decrease of 26.62%, and the content of the relevant substances exceeded 2 months later. The area of the main peak of the control solution did not meet the requirements, indicating that its stability was poor. It can be seen that the disaccharide can well protect the trace tetrodotoxin.
  • Example 2 A freeze-dried formulation containing 30 ⁇ of tetrodotoxin and 30 mg of dextran was prepared. According to the method described in Example 1, a white sparse muffin can be obtained by freeze-drying the solution with a pH value of 4.0 and containing 0.003% tetrodotoxin and 3% dextran, and the preparation is stabilized at 40 ° C. In the study, samples were taken on days 1, 3, 5, and 10, and the tetrodotoxin content and related substances were determined by HPLC. The results were compared with the results obtained on day 0. The results are shown in Table 10. Table 10
  • dissolved water-soluble solvent dissolves to produce a sterile, pyrogen-free clear solution that can be directly administered for intramuscular or subcutaneous injection.
  • a white muffin-like substance can be obtained by freeze-drying a solution having a pH value of 6.0 and containing 0.00005% tetrodotoxin and 10% lactose, which can be regenerated with a water-soluble solvent, and can be directly supplied to muscles or Subcutaneous injection.
  • Example 5 Frozen prepared containing the biologically active substance and tetrodotoxin 5 ⁇ ⁇ 15mg lactose, sucrose, 15mg (or maltose, cellobiose) in Formulation Example was dry.
  • a white muffin-like substance can be obtained by freeze-drying a solution having a pH value of 4.5 and containing 0.0005% tetrodotoxin, 1.5% lactose, and 1.5% sucrose, and the preparation is sterilized.
  • Water for injection or a pharmaceutically acceptable water-soluble solvent dissolves to produce a sterile, pyrogen-free clear solution that can be directly administered for intramuscular or subcutaneous injection.
  • Example 6 A freeze-dried preparation containing 20 ⁇ ⁇ of tetrodotoxin and 15 mg of lactose (or sucrose, maltose, and cellobiose) and 15 mg of mannitol was prepared. According to the method described in Example 1, a white muffin-like substance can be obtained by freeze-drying a solution having a pH value of 5.5 and containing 0.002% tetrodotoxin, 1.5% lactose and 1.5% mannitol, and the preparation is sterilized. Water for injection or a pharmaceutically acceptable water-soluble solvent dissolves to produce a sterile, pyrogen-free clear solution that can be directly administered for intramuscular or subcutaneous injection.
  • the content of tetrodotoxin is 99.65%, and the content of related substances is less than the area of the main peak of the control solution, which meets the requirements for clinical use.
  • this purpose cannot be achieved when only mannitol is used as an excipient, thereby fully explaining that lactose or sucrose, maltose, cellobiose and the like have obvious protective effects in the lyophilized preparation.
  • Industrial Applicability The lyophilized preparation according to the present invention can be used as a pharmaceutical preparation.

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稳定的医药用河豚毒素冷冻干燥制剂 技术领域 本发明涉及一种医药用冻干制剂， 特别是稳定的含有符合人体安全注射使用的河豚毒 素的冷冻干燥制剂。 背景技术 河豚毒素是自然界中发现的非蛋白质神经毒素之一， 可高选择和高亲和性地与神经细 胞膜钠离子通道 SS1/SS2结合， 几十年来一直在神经药理学和肌肉生理学等研究领域中被 广泛用作药理学工具药，市场上典型产品为 Sigma-Aldrich公司提供的 1毫克河豚毒素含量 的柠檬酸盐固态冻干粉 （该公司货号: T5881 )。 河豚毒素除了用于科研之外， 1998年起， 发明人所在公司己申报河豚毒素注射液 （水 溶液） 为一类新药， 用于戒毒、 镇痛等治疗用途 (Pan, et al. US 5,846,975; Dong, et al. US 6,407,088) 但河豚毒素注射液 （水溶液） 对温度很敏感， 容易受温度的影响而发生降解， 温度越 高， 降解越快。 一旦有效活性物质河豚毒素含量低于其标示量的 90%或有关物质的相对含 量超过医药标准规定限度（大于对照溶液主峰面积）就不符合临床使用要求。我们用 HPLC 测定了河豚毒素注射液中河豚毒素的含量及有关物质的相对含量， 结果发现， 河豚毒素注 射液的含量随温度和贮存时间而变化， 试验结果表明： 40°C条件下， 1 天后河豚毒素的含 量从 0天的 100%降为 91.9%， 下降了 8.1%， 3天后降为 89.37%， 下降了 10.63% (见表 1 )。 以上结果表明， 在 4CTC条件下， 河豚毒素注射液在放置 3天后， 其含量低于规定限度， 不 符合药用要求。 在 25°C条件下， 河豚毒素注射液在放置 1个月后， 其含量由 0月的 100% 降为 95.34%， 下降了 4.66%， 有关物质含量大于对照溶液主峰面积， 超过规定限度， 不符 合医药用标准要求； 在放置 3个月后， 河豚毒素的含量降为 89.77%， 下降了 10.23%， 有关 物质含量大于对照溶液主峰面积， 两者均不符合医药用标准要求 （见表 2)。 以上结果表明， 河豚毒素注射液在 25 °C下贮存， 不能保证其产品质量， 1个月后， 有 关物质含量就超过规定， 不符合医药用标准要求。 为了保证河豚毒素注射液的质量， 防止 河豚毒素含量下降和有关物质含量升高， 必须将其置于 4一 8°C冰箱中保存。这就给河豚毒 素的临床使用带来诸多困难和不便， 相应在贮存、 运输、 装卸、 批发、 零售、 医院和应用 各个环节都得注意保持 4-8 °C低温， 否则温度偏高可能影响临床效果，对此， 我们必须设法 尽快予以解决， 研制出安全稳定， 室温下就能长期贮存的可靠产品。 表 1 河豚毒素注射液 （990120A) 40 °C稳定性研究
试验条件 贮藏时间 备 注
0 天 无色澄明液体 100 以 0天含量
1 天 无色澄明液体 91.9 作 100%计。
40 °C. 3 天 无色澄明液体 89.37
5 天 无色澄明液体 87.45
10 天 无色澄明液体 88.06
说明： 含量 <90%即不符合标准规定。 表 2 河豚毒素注射液 25°C稳定性研究
时间 （月） 含 量 （％) 有关物质 （《 备 注
0 100 <对照溶液主峰面积 以 0天含量作 i00%
1 95. 34 >对照溶液主峰面积 "^。
2 93. 72 >对照溶液主峰面积
3 89. 77 >对照溶液主峰面积
6 82. 47 〉对照溶液主峰面积
说明： 含量 <90%或有关物质 >对照溶液主峰面积， 即不符合标准规定。 试验证明， 河豚毒素注射液 （水针剂） 在室温下不稳定， 必须将其在低温下贮藏 （4 一 8°C )， 这给运输、 贮存、 使用带来不便。 为了解决上述问题， 一个简单的做法是将河豚 毒素做成冻干制剂。 一般在水溶液中不稳定的生物活性物质在冻干脱水后贮存期都可延 长， 待临床应用时， 加灭菌注射用水再生即可。 但河豚毒素用于医药时， 其每剂量的用量 只有 0.5〜60μ2， 如此微量的河豚毒素在溶液中经冻干脱水后形成不了所要求的固态残留 物，必需加入医药上能接受的赋形剂， 才能提供微量河豚毒素得以附着的支架， 在溶液冻干 脱水后能形成固状物。 然而我们不论用河豚毒素工具药中所釆用的柠檬酸盐或是最常用的甘露糖醇来作赋 形剂， 实验结果表明： 所制成河豚毒素的冻干制剂在 40°C贮存条件下， 用柠檬酸盐作赋形 剂时， 产品的外观会收缩变形， 不符合医药用规定要求， 河豚毒素含量会逐渐下降， 有关 物质含量在 5天后大于对照溶液主峰面积， 超过规定限度， 不符合医药用标准要求； 用甘 露糖醇作赋形剂时， 产品外观虽符合规定， 但含量会逐渐下降， 有关物质含量在 5天后也 大于对照溶液主峰面积， 超过规定限度， 不符合药用要求 （见表 3)。 在 25°C条件下贮存 6 个月后，选用柠檬酸盐作赋形剂，河豚毒素的含量由 0月的 100%降为 95.1%，下降了 4.9%， 有关物质含量大于对照溶液主峰面积， 超过规定限度， 不符合医药用标准要求； 选用甘露 糖醇作赋形剂， 6个月后， 河豚毒素的含量由 0月的 100%降为 96.59%, 下降了 3.41%， 有 关物质含量大于对照溶液主峰面积， 超过规定限度， 也不符合医药用标准要求 （见表 4)。 配方 柠檬酸盐 甘露糖醇 配方 柠檬酸盐 甘露糖醇 白色疏松饼状物， 40'C下 白色疏松饼状物, 40'C下 白色疏松饼状物，但会慢 白色疏松饼状物，室温下 外观 放置 1天几乎完全收缩变 放 S 10 天未见明显变 外观 慢收缩变形成晶状物附 贮存未见明显变化,外观 形， 外观不符合规定。 化， 外观符合规定。 于瓶壁， 外观不合格。 符合规定。
微 有关物质 含 fl 有关物质 微 含 fl 有关物质 含 fi 有关物质 含 fl(%)
m (%) (%) (%) 纖质 (%) (%) (%) (%)
<对照溶液 <对照溶液 <对照溶液 <对照溶液 限度 90-110 90-110 限度 90-1 10 90-110
主峰面积 主峰面积 主峰面积 主峰面积
<对照港液 <对照溶液 <对照溶液 <对照溶液
0天 100 100 0月 100 100
主峰面积 主峰面积 主峰面积 主峰面积
<对照溶液 <对照溶液 <对照溶液 <对照溶液
1天 98.97 99.86 1月 98.8 99.69
主峰面积 主峰面积 主峰面积 主峰面积
<对照溶液 <对照溶液 <对照溶液 <对照溶液
3天 97.26 99 2月 97.2 98.14
主峰面积 主峰面积 主峰面积 主峰面积
>对照溶液 >对照溶液 <对照溶液 <对照溶液
5天 96.19 97.69 3月 96.6 98.04
主峰面积 主峰面积 主峰面积 主峰面积
>对照溶液 >对照溶液 >对照溶液 >对照溶液
10天 93.18 95.55 6月 95.1 96.59
主峰面积 主峰面积 主峰面积 主峰面积 说明： 含量 <90%或有关物质 >对照溶液主峰面积， 即不符合标准规定， 不能供医药用。 这提示我们： 现有技术的冻干制剂中， 赋形剂只起到物理上的部份稳定作用， 我们检 索了所有公开文献， 未发现有介绍在微量河豚毒素冻干粉剂制备时能增加化学稳定性的添 加剂。 我们逐渐意识到必须从河豚毒素的化学结构着手， 开辟一条寻求提高河豚毒素实用 制剂对温度稳定的新途径。 河豚毒素的化学名为 10氢 -12- (径甲基) -2-亚氨基 -5，9:7， 10 α -二甲桥 -10 α Η-(1,3) 二桥 氧并 (6，5-d)-嘧啶 -4,7,10,11， 12-戊醇， 分子式为 CuH17N308， 分子量为 319.28， 结构式如下：
Figure imgf000004_0001
河豚毒素的结构式 河豚毒素在 220°C以上变黑， 但不分解， [ a ] ^5 -8.64 (C=8.55 稀醋酸中）。 PKa 8.76 (水）； 9.4 (50%乙醇 溶于稀醋酸， 微溶于水、 无水乙醇、 乙醚， 不溶于其他有机溶剂， 在强 酸和强碱溶液中毒性破坏。 （The Merck Index. 13th Ed. 2001, 9318) 河豚毒素在固态时对热是相当稳定的， 但是它的水溶液特别是低浓度的稀酸水溶液稳 定性较差 （Kang， et al. US 6,559,154) 技术内容 本发明的目的在于提供一种河豚毒素或其类似物的稳定的冷冻干燥制剂。 为了达到上述发明目的， 本发明提供了一种稳定的医药用冷冻干燥制剂， 其包含作为 生物活性物质的河豚毒素或其类似物，所述的河豚毒素或其类似物的含量为 0.5-60μ§/每剂 量， 所述的制剂还包含能够降低河豚毒素或其类似物 C-4羟基活泼性的化合物， 优选为双 糖、 聚糖或其衍生物之任一种或其混合物的含有糖苷键的化合物。 具体而言， 为了获得上述的技术方案， 本申请的发明人对一下问题进行了深入的研究 和探讨：
①河豚毒素在酸性水溶液中含量为什么会下降？
②河豚毒素含量下降后其转化产物是什么？转化产物与河豚毒素比较有什么差别？
③河豚毒素的转化机理？
④如何阻止河豚毒素的转化？ 对问题①： 河豚毒素在酸性水溶液中含量为什么会下降？
1965年 T.GOTO等人就指出， 在酸的催化作用下河豚毒素发生差向异构化， 转变为 4- 表河豚毒素， 然后再转变为 4,9-脱水河豚毒素 （Tetrahedron. 1965. Vol.21. 2059-2088), 0 此 夕卜， 河豚毒素在水中加热迴流时会变为河豚酸 (Annals New York Academy of Sciences. 1985, 479:32-43) 下式表示河豚毒素和 4-表河豚毒素、 4,9-脱水河豚毒素以及河豚酸之间的转化。
Figure imgf000006_0001
Figure imgf000006_0002
河豚毒素及其衍生物之间相互转化方式 由于河豚毒素在酸性水溶液中会发生差向异构化而转化为 4-表河豚毒素和 4,9-脱水河 豚毒素， 从而导致含量下降。 若加热回流， 则直接变为河豚酸。 本申请人长期对河豚毒素的提取、 纯化、 结构改造及异构化后的产物 4-表河豚毒素和 4,9-脱水河豚毒素的测定等研究， 证实了以上的结论。 对问题②： 河豚毒素含量下降后其转化产物是什么？ 转化产物与河豚毒素有何差别？ 文献报导河豚毒素在酸性水溶液中会转化为 4-表河豚毒素和 4，9-脱水河豚毒素， 为确 证这一结论， 我们进行了河豚毒素在水溶液中的热动力学研究。 研究进行如下： 取一定浓度的 (百分浓度)不同 pH值的河豚毒素溶液， 分别在 80°C ± 1 °C和 90°C ± C水溶液中平衡，然后在不同的平衡时间点取样并立即冷冻到 -18Ό，用 HPLC 测定转化产物及其含量(∑110^ 61 &1. 118. 6,562，968)， 结果见表 5、 表 6。 80 °C下河豚毒素热动力学研究 (溶液 pH值 4.67)
Figure imgf000007_0002
由表 6可见， 河豚毒素在 90°C水溶液中会转化为 4-表河豚毒素和 4,9-脱水河豚毒素， 并有部分转化为河豚酸。
Figure imgf000007_0001
(1 ) 4· 可豚 Si ¾ (2) 河腺继紫 (3) 河豚酸
河豚毒素的常见衍生物
( 1 ) 4-表河豚毒素 （2) 4,9-脱水河豚毒素 （3 ) 河豚酸 通过以上研究， 我们确认了河豚毒素在水溶液会转化为 4-表河豚毒素和 4,9-脱水河豚 毒素， 如果温度达到 90°C , 河豚毒素就会有部分转化为河豚酸。 河豚毒素、 4-表河豚毒素、 4,9-脱水河豚毒素在化学上非常类似， 4-表河豚毒素与河豚 毒素的分子式、 分子量完全相同， 唯一区别是 4位羟基的立体位置不同； 4,9-脱水河豚毒 素与河豚毒素和 4-表河豚毒素的分子式只差一个 H20， 分子量少 18， 但它们的生物活性却 有显著差异。 例如河豚毒素的毒性为 4500鼠单位 /毫克， 4-表河豚毒素的毒性为 710鼠单 位 /毫克， 4,9-脱水河豚毒素的毒性只有 92鼠单位 /毫克 (Toxicon. 1985, 23:271〜276), 而河 豚酸的毒性只有河豚毒素的约 1/700。 鉴于河豚毒素、 4-表河豚毒素和 4,9-脱水河豚毒素的生物活性差异极为显著， 故河豚 毒素一旦转化为 4-表河豚毒素和 4,9-脱水河豚毒素后， 其原有的生物活性会大大下降， 并 失去其医药用价值。 问题③： 河豚毒素转化为 4-表河豚毒素和 4,9-脱水河豚毒素的化学机理？ 从河豚毒素的分子结构上很容易看出 C-4 的特殊性， 联有平伏键羟基和直立键氢原子 的 C-4在氮的邻位， 因此， C-4上的羟基具有特殊的化学和生理活性。 当溶液中有 H+存在， H+将会与 C-4羟基上的氧原子结合，由结构 A变成带正电荷的结构 B，结构 B失去一分子水， 生成 C-4带正电荷的结构。。 在溶液中结构 C与水分子结合， 水分子既可从原来脱去水分子 的位置上进行攻击而衍生出结构 E, 也可从脱去水分子位置的反面进攻， 而衍生出结构 D。 从结构 E除去 H+， 则又产生原来的河豚毒素即结构 A， 从结构 D除去 H+， 则会产生结构 F。 结构 F和结构 A之间的区别在于氢原子和羟基的位置互相交换，在结构 A中， C-4的 H原子是 直立键， 羟基是平伏键， 而在结构 F中， C-4上的 H原子是平伏键， 而羟基是直立键。 结构 A和结构 F两者在化学上称为差向异构体。 河豚毒素发生异构化的机理如下：
Figure imgf000008_0001
A 河豚毒素 4位羟基异构化的机理 结构 A称作 "河豚毒素"， 是自然界中从河豚鱼得到的河豚毒素 （TTX) 的主要成分。 而结构 F通常称为 4-表河豚毒素。 由于其 C-4上的羟基与 C-9上的羟基很接近，在 H+的作用 。下，很容易失去一分子水而生成更加稳定的 4,9-脱水河豚毒素。这三种所谓的"河豚毒素"， 其化学性质只有稍微差别， 而其生物活性却有明显不同。 从以上的分析，我们可以得出结论，河豚毒素的不稳定性是由于水分子的作用，在 H+的 协助下发生差向异构化， 差向异构化后进一步导致 4,9-脱水河豚毒素的生成。 为了阻止河 豚毒素 的差向异构化， 就是要阻止 4位碳上的羟基异构化， 当然想彻底改变河豚毒素的 稳定性， 最好的办法是把 C-4上的羟基还原， 为此， 我们从河豚毒素合成了 4-去氧河豚毒 素， 其结构式如下：
0一
Figure imgf000009_0001
4-去氧河豚毒素
4-去氧河豚毒素的结构式
4-去氧河豚毒素非常稳定， HPLC分析显示其水溶液加热煮沸 2h后没有转变， 但是其 11)5()为 3336.5 g kg， 毒性比河豚毒素要小 330倍。进一步用小鼠醋酸扭体法测定它的镇痛 效果,结果显示其镇痛效果也比河豚毒素小约 330倍。 从上述所有实验结果我们得出结论:河豚毒素分子中 4位碳原子上的羟基是活性中心， 它的存在对河豚毒素分子的生物活性起关键作用， 而使 C-4羟基保持在水平位置又是它的 稳定的关键所在。 促使它转变为直立位置的是水分子， 这就是问题④如何阻止河豚毒素的 转化之所在， 即我们筛选医药用微量河豚毒素冻干制剂的配方时， 应寻找能使 C-4羟基保 持在水平位置上的辅料， 以增强河豚毒素在制剂中的稳定性。 由此， 发明人摸索了一种解决上述问题的方法。 由于 C- 4羟基的立体位置是河豚毒素具有生物活性的关键基团之一，本申请的发明人 以此为出发点， 寻找一种既能为医药上可接受又不改变 C- 4羟基的性质与立体位置， 从而 阻止其发生差向异构化的物质为辅料。 首先考虑羟 基上的氢原子能与电负性很大的原子 如氧原子形成氢键。 因氧原子外层有 6个电子， 在形成化合物后， 仍有两对未用电子对， 故电负性很大， 很容易与 C- 4羟基上的氢原子和氮原子上的氢原子形成氢键， 这样通过氢 键的形成就生成了一个六元环的化合物， 从而 "锁定 " 了 C- 4上的羟基， 也就是使 C- 4羟 基立体空间位置被固定。 其次考虑河豚毒素结构上两个六元环是两个椅式结构拼合而成， 根据相似兼容法则， 能否找到一个化合物与河豚毒素结构类似， 能将河豚毒素分子包围起 来。 能符合以上两种要求的化合物为含糖苷键的化合物如双糖等化合物。 鉴于以上分析及 不断研究， 我们研究发现在河豚毒素制剂中加入一定量的双糖如乳糖、 蔗糖、 纤维二糖、 麦芽糖， 通过冷冻干燥后制成的冻干剂， 确实解决了河豚毒素在制剂中的稳定性， 室温下 贮存 1年后，其河豚毒素的含量及有关物质含量均未发生明显变化，符合医药用规定标准 < 以下以乳糖为例描述河豚毒素和双糖通过形成氢键而阻止 C- 4羟基异构化的机理。
Figure imgf000010_0001
河豚毒素 乳糖
Figure imgf000010_0002
基于同一原理， 聚糖如缩合葡萄糖、 右旋糖酐等及其衍生物如羟乙基淀粉、 羟丙基环 糊精等分子中也含有糠苷键， 且分子结构与河豚毒素的结构有相似之处， 也应对河豚毒素 有稳定作用。我们研究发现聚糖确实有此作用。而分子中没有糖苷键的化合物，如单糖（葡 萄糖、 果糖、 甘露糖等） 则不能 "锁定" C- 4上羟基的空间位置， 故不能阻止河豚毒素差 向异构化的发生， 其原因可能是由于在单糖中氧原子在环中， β异头物中存在 C- 1羟基偶 极和环氧偶极之间平行的相互作用， 它们之间相互排斥， 这种排斥作用不利于环氧原子形 成六元环的氢键（王镜岩等. 生物化学. 北京:高等教育出版社， 2002. 13)。
Figure imgf000010_0003
平 伏羟基 (β异头物) 单糖 β异头物环氧原子的偶极与 C-1上羟基的偶极互相平行 本发明的技术方案如下- 为了克服现存技术的不足之处， 本发明人发明了一种以冻干制剂形式提供原在水溶液 或柠檬酸盐冻干形态都不够稳定的生物活性成分一河豚毒素的医药配方及制法， 从而达到 室温下就能长期贮存的目的。 临床应用时加入人体可接受的水溶液载体后即可再生， 供注 射给药。 本发明所述的稳定的医药用河豚毒素冷冻干燥制剂， 是含有微量生物活性物质河豚毒 素 （Tetrodotoxin TTX)、 稳定剂、 助溶剂的药物组合物， 所述的河豚毒素在冷冻干燥制 剂组合物中的量仅为 0.5-60μβ/每剂量，其余为稳定剂、 助溶剂。 以上所述河豚毒素包括河豚毒素和其类似物脱水河豚毒素， 胺基河豚毒素， 甲氧基河 豚毒素， 乙氧基河豚毒素， 具有生物活性成分的河豚毒素是从海洋动物河豚鱼的卵巢、 肝 脏中提取得到， 或者从其它物种， 如两栖类动物、 扁形动物涡虫类、 纽形动物的纽虫类、 棘皮动物海星类、 毛颚动物箭虫类、 软件动物腹足类等以及某些细菌如溶藻弧菌等细菌中 提取得到。 提取方法可参照现有公开文献的方法， 例如本申请人的专利 (Zhou,et al., US 6,552,191)。 其类似物是用河豚毒素经结构改造而得到的化合物。 在本发明中， 作为符合人体安全注射使用的生物活性物质的河豚毒素， 其含量极微， 每剂量只有 0. 5μβ〜60μδ， 无法制成冻干制剂， 通常必须加入医药上可接受的赋形剂， 以 增加冻干前溶液的浓度。 研究发现， 只有加入一种或一种以上对河豚毒素有稳定作用的物 质才能阻止河豚毒素在贮存期间发生差向异构化， 阻止其转变为 4-表河豚毒素和 4， 9 -脱 水河豚毒素。 在本发明中， 能阻止河豚毒素差向异构化并具有稳定作用的物质是乳糖、 蔗 糖、 麦芽糖和纤维二糖等双糖或缩合葡萄糖、 右旋糖酐等聚糖或其衍生物如羟乙基淀粉、 羟丙基环糊精等， 可以单独使用或两种以上混合使用， 每剂量的用量为 5mg〜100mg。 本发明中， 生物活性物质河豚毒素几乎不溶于水， 为了获得所需浓度的水溶液， 必须 加入一种能增加河豚毒素溶解度的助溶剂。从河豚毒素的化学结构分析，它是一个有机碱， 故可溶于酸中， 但强酸会使其分解， 经过研究， 只有加入不挥发的有机酸如柠檬酸、 苹果 酸、 酒石酸、 乳糖酸等最理想， 它们能与河豚毒素形成有机酸盐而溶于水中， 又容易控制 其酸度。 试验证明， 本发明中所用助溶剂的量应使冻干前溶液的 pH值为 3. 0〜6. 0， 通常 每剂量用量为 0. 00005〜0. 0005mg。
- 本发明是釆用冷冻干燥法将含生物活性物质河豚毒素和双糖或聚糖或其衍生物的水 溶液或水溶性溶剂的溶液经冷冻干燥后而得到固体 （Reminton's Pharmaceutical Sciences. Seventeenth Edition. 1985, 1314. ) o所述的水溶液很容易制备， 通过 0. 22μηι的膜， 再通过截 留分子量为 10000的超滤器， 经冷冻干燥后即可得到无菌和无热原的冻干制剂， 如此得到 的冻干制剂是稳定的， 能较长时间阻止生物活性物质河豚毒素的转化。 为了使得到的冻干制剂有一个恒定合适的 pH值， 并避免注射时引起局部组织的刺激 疼痛或坏死， 通常加入计算量的助溶剂， 使 pH值控制在 3. 0〜6. 0之间， 如果 pH值低于 3. 0, 可用稀氢氧化钠 （钾）溶液或相应助溶剂的钠 （钾） 盐进行调节。 其用量应根据冷 冻干燥前溶液的 pH值而定， 如果溶液的 pH值已在 3. 0〜6. 0之间， 则可以不必调节 pH 值。 本发明制备含微量河豚毒素冷冻干燥制剂的方法是： 将助溶剂、 稳定剂和 pH值调节 剂分别溶解于水中， 将微量河豚毒素溶于计量的助溶剂中， 再加到稳定剂溶液中， 加水至 规定体积， 混合均匀， 测定 pH值应在 3. 0〜6. 0之间， 否则用氢氧化钠 （钾） 或相应的有 机酸钠 （钾） 盐调节 pH值， 经除菌过滤、 超滤后定量灌装于玻璃小瓶中， 半加塞、 冷冻 干燥、 压塞、 轧盖即得。 以上所述的冷冻干燥制剂组合物用人体可接受的水溶液载体再生， 得到可用于肌肉或 皮下注射给药的无菌、 无热原澄明液体， 水溶液载体为灭菌注射用水或其它水溶液载体， 水溶液载体的用量为 0.5— 5ml， 最佳用量为 1一 2ml。 具体制备工艺是-
1. 将定量助溶剂溶于注射用水中。
2. 将 pH值调节剂溶于注射用水中。
3. 将稳定剂双糖或聚糖或其衍生物溶于注射用水中。
4. 向微量河豚毒素中加入计算量的助溶剂溶液， 搅拌， 使溶解。
5. 将 4力口入 3中， 并加注射用水至规定体积， 摇匀。
6. 测定溶液的 PH值， 应在 3. 0〜6. 0之间， 否则， 用 pH值调节剂溶液调节。
7. 除菌过滤（例如 Millipore公司的过滤系统）和超滤（例如 Pull公司的超滤系统） 后得到无菌、 无热原的澄明溶液。
8. 将 7得到的溶液在无菌条件下定量分装于玻璃小瓶中， 半加塞后， 送至冷冻干燥 机中， 预冻至物品温度达到 -4CTC以下， 打开冷阱制冷， 使冷阱温度下降到 -50Ό以 下， 起动真空泵， 维持真空度在 5帕以下， 并自然升温至设定值， 维持 24小时， 然后升温至 30Ό维持 10小时以上， 自动压塞。
9 -取出轧盖即得。 与现有技术相比， 本发明突出的实质性和显著的进步是- 河豚毒素注射液无论在冷藏（4-8°C )或常温下都非常难以保存，特别在常温条件下很 容易发生差向异构化， 转变为 4-表河豚毒素和 4，9-脱水河豚毒素而失去药用价值， 本发明 人正是经过大量的研究， 找到了能阻止河豚毒素差向异构化的稳定剂， 使得制剂中极微量 的河豚毒素能在常温下保持稳定。 具体的优点如下：
1. 制剂配方中加入稳定剂双糖或聚糖或其衍生物， 能有效保护河豚毒素在贮存期间 不发生差向异构化， 能有效阻止河豚毒素转化为 4-表河豚毒素和 4， 9-脱水河豚 毒素， 产品质量得到保证。
2. 采用冷冻干燥技术， 产品中含水份很少， 提高了产品的稳定性。
π 3. 以上两种技术的结合， 能有效解决医药用微量河豚毒素在制剂中的稳定性。
4.产品不需在 4—8°C冰箱中保存， 可在常温下保存， 大大节约贮藏、 运输成本， 方便 医疗使用，更重要的是提供了安全可靠、 质量稳定、 室温下贮存期可长达壹年以上的 产品。 本发明的最佳实施方式 实施例 1 以表 7所示的量制备河豚毒素与双糖（乳糖、 蔗糖、 麦芽糖、 纤维二糖） 的冷冻干燥 制剂。 配方 1用果糖 （单糖） 作赋形剂， 配方 2、 3、 4、 5用双糖作稳定剂（同时兼作赋 形剂）。 将柠檬酸 （助溶剂）和稳定剂分别溶于注射用水中， 将河豚毒素溶于柠檬酸溶液 中， 后将稳定剂溶液加入其中， 加注射用水至规定体积， 搅拌均匀， 调节 pH值为 4.0， 除 菌过滤、 超滤， 定量灌装于玻璃小瓶中， 半加塞， 送入冷冻干燥机中， 预冻至物品温度达 到 -40Ό以下， 打开冷阱制冷， 使冷阱温度下降到 -50°C以下， 起动真空泵， 维持真空度在 5 帕以下，并自然升温至设定值，至少 24小时，再自然升温至 30°C至少维持 10小时，压塞、 轧盖即得。 表 7
Figure imgf000013_0001
将以上所得到的河豚毒素冻干制剂和河豚毒素注射液（水针剂）在 4(TC高温下同时进 行稳定性试验， 于 1、 3、 5、 10天取样， HPLC法测定河豚毒素含量和有关物质含量， 并 与 0天测定结果比较， 结果见表 8。 不同配方河豚毒素冻干剂与河豚毒素注射液 40Ό高温下稳定性试验结果
Figure imgf000014_0001
说明： 1. 6* 为河豚毒素注射液（水针剂)
2. 含量 <90%或有关物质 >对照溶液主峰面积， 即不符合标准规定， 不能供医药用。
配方 1用果糖作赋形剂， 产品外观不符合要求， 在 40°C高温试验下， 河豚毒素的含量 会逐渐下降， 由 100% (0天） 降为 95.99% ( 10天）， 下降了 4.01%, 有关物质含量在 5天 后即超过对照溶液主峰面积， 不符合规定要求， 故果糖不能阻止河豚毒素的差向异构化的 发生， 不能解决河豚毒素的稳定性。 配方 2、 3、 4、 5 分别用乳糖、 蔗糖、 麦芽糖及纤维 二糖等双糖作稳定剂时， 在 40Ό高温试验条件下， 10天后河豚毒素的含量及有关物质含 量均未发生明显变化， 河豚毒素的含量分别为 99.97%、 99.56%、 99.18%和 99.75%， 有关 物质含量均小于对照溶液主峰面积， 符合医药用标准要求， 稳定性大大提高， 说明双糖能 阻止河豚毒素的差向异构化， 达到了保护河豚毒素的目的。 配方 6*为水针剂， 在同样试验 条件下， 河豚毒素的含量逐渐下降， 由 100% (0天）降为 90.06% ( 10天）， 下降了 9.94%， 有关物质含量自第 3天起即超过对照溶液主峰面积， 不符合规定要求。 由此说明河豚毒素 冻干剂采用双糖作稳定剂后， 稳定性比水针剂有了明显提高。 将以上得到的河豚毒素冻干剂和河豚毒素注射液 （水针剂） 在室温下同时进行长期稳 定性试验， 于 1、 2、 3、 6、 9、 12个月取样， HPLC法测定河豚毒素含量和有关物质含量， 并与 0月测定结果比较， 结果见表 9。
不同配方河豚毒素冻千剂与河豚毒素注射液室温下稳定性试验结果
Figure imgf000016_0001
说明： 1. 6* 为河豚毒素注射液 （水针剂）
2. 含量 <90%或有关物质 >对照溶液主峰面积， 即不符合标准规定， 不能供医药用。
配方 1用果糖作赋形剂， 制剂外观不合格， 贮存期间河豚毒素的含量下降， 12个月后 含量由 100% (0月） 降为 83.19%， 下降了 16.81%， 2个月后有关物质含量已超过对照溶 液主峰面积， 不符合规定要求， 不能供医药用。 配方 2、 3、 4、 5分别用乳糖、 蔗糖、 麦 芽糖及纤维二糖等双糖作稳定剂， 在室温下放置 12个月， 其外观、 河豚毒素的含量和有 关物质含量均未发生明显变化， 河豚毒素的含量分别为 101.92%、 99.89%, 99.87%和 99.47%,有关物质含量均小于对照溶液主峰面积，符合医药用标准要求。配方 6A为水针剂， 在同样条件下贮存， 河豚毒素含量下降明显， 12个月后由 100% (0月） 降为 73.38%， 下 降了 26.62%， 有关物质含量自 2个月后即超过对照溶液主峰面积， 不符合规定要求， 说明 其稳定性较差。 可见双糖能很好地保护微量河豚毒素， 室温下贮存 12个月后， 其河豚毒 素的含量和有关物质含量仍符合临床应用要求， 解决了河豚毒素制剂的稳定性。 实施例 2 制备含生物活性物质河豚毒素 30μ 和 30mg右旋糖酐的冷冻干燥制剂。 按实施例 1 所述的方法， 将 pH值为 4.0， 内含 0.003%河豚毒素和 3%右旋糖酐溶液经冷冻干燥， 即可 得到白色疏松饼状物， 将该制剂用在 40°C进行稳定性研究， 于 1、 3、 5、 10天取样， HPLC 法测定河豚毒素含量和有关物质含量， 并与 0天测定结果比较， 结果见表 10。 表 10
Figure imgf000017_0001
以上结果表明， 右旋糖酐因含有和双糖类似的化学结构， 故在配方中对河豚毒素有稳定作 用， 在 40°C高温条件下， 其外观、 含量及有关物质均符合标准规定。 实施例 3 制备含生物活性物质河豚毒素 60μ§和 5mg乳糖 （或蔗糖、 麦芽糖、 纤维二糖） 的冷 冻干燥制剂。 按实施例 1所述的方法， 将 pH值为 3.0, 内含 0.006%河豚毒素和 0.5%乳糖 的溶液经冷冻干燥， 即可得到白色疏松饼状物， 将该制剂用灭菌注射用水或医药上可接收 的水溶性溶媒溶解， 生成无菌、 无热原的澄明溶液， 可直接供肌肉或皮下注射给药。 实施例 4 制备含生物活性物质河豚毒素 0.5μ§和 lOOmg乳糖（或蔗糖、 麦芽糖、 纤维二糖） 的 冷冻干燥制剂。 按实施例 1所述的方法， 将 pH值为 6.0， 内含 0.00005%河豚毒素和 10% 乳糖的溶液经冷冻干燥， 即可得到白色疏松饼状物， 加水溶性溶媒再生， 可直接供肌肉或 皮下注射给药。 实施例 5 制备含生物活性物质河豚毒素 5μ§和 15mg乳糖、 15mg蔗糖（或麦芽糖、纤维二糖) 的冷冻千燥制剂。按实施例 1所述的方法，将 pH值为 4.5， 内含 0.0005%河豚毒素和 1.5% 乳糖、 1.5%蔗糖的溶液经冷冻干燥， 即可得到白色疏松饼状物， 将该制剂用灭菌注射用水 或医药上可接收的水溶性溶媒溶解， 生成无菌、 无热原的澄明溶液， 可直接供肌肉或皮下 注射给药。 实施例 6 制备含生物活性物质河豚毒素 20μ§和 15mg乳糖 （或蔗糖、 麦芽糖、 纤维二糖） 与 15mg甘露糖醇的冷冻干燥制剂。 按实施例 1所述的方法， 将 pH值为 5.5， 内含 0.002%河 豚毒素和 1.5%乳糖及 1.5%甘露醇的溶液经冷冻干燥即可得到白色疏松饼状物， 将该制剂 用灭菌注射用水或医药上可接收的水溶性溶媒溶解， 生成无菌、 无热原的澄明溶液， 可直 接供肌肉或皮下注射给药。 按本实施例得到的样品， 在室温上贮存一年后， 其河豚毒素含量为 99.65%， 有关物质 含量小于对照溶液主峰面积， 符合临床使用要求。 而仅用甘露糖醇作赋形剂时则不能达到 此目的， 由此更充分说明乳糖或蔗糖、 麦芽糖、 纤维二糖等在本冻干制剂具有明显的保护 作用。 工业应用性 按照本发明的冷冻干燥制剂可以用作医药用制剂。

Claims

权利要求
1. 一种稳定的医药用冷冻干燥制剂， 其包含作为生物活性物质的河豚毒素或其类似 物， 其特征在于， 所述的河豚毒素或其类似物的含量为 0.5-60μ§/每剂量， 所述的制剂还包 含能够降低河豚毒素或其类似物 C-4羟基活泼性的化合物。
2. 如权利要求 1所述的制剂， 其中， 所述的能够降低河豚毒素或其类似物 C-4羟基 活泼性的化合物为含有糖苷键的化合物。
3. 如权利要求 2所述的制剂， 其中， 所述的含有糖苷键的化合物为双糖、 聚糖或其 衍生物之任一种或其混合物。
4. 如权利要求 1一 3之一所述的制剂， 其中， 所述的河豚毒素或其类似物选自河豚毒 素， 脱水河豚毒素， 氨基河豚毒素， 甲氧基河豚毒素， 乙氧基河豚毒素所组成的组。
5. 如权利要求 3所述的制剂， 其中， 所述的含有糖苷键的化合物的量为 5-100mg/每 剂量。
6. 如权利要求 3 所述的制剂， 其中， 所述的双糖选自由乳糖、 蔗糖、 麦芽糖、 纤维 二糖组成的组； 所述的聚糖为缩合葡萄糖或右旋糖酐， 其衍生物为羟乙基淀粉或羟丙基环 糊精。
7. 如权利要求 1 所述的制剂， 其中， 所述的制剂还包含选自柠檬酸、 酒石酸、 苹果 酸、 乳糖酸或其组合的助溶剂， 所述助溶剂的用量使得在制备所述冻干制剂前溶液的 pH 值在 3.0〜6.0之间。
8. 如权利要求 7所述的制剂， 其中， 所述的助溶剂的量为 0.00005-0.0005mg/每剂量。
9. 如权利要求 1所述的制剂， 其中， 所述的生物活性物质是河豚毒素。
10. 如权利要求 1所述的制剂， 其中， 所述的制剂还包含医药上可接受的赋形剂。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010031346A1 (zh) * 2008-09-17 2010-03-25 厦门朝阳生物工程有限公司 河豚毒素冻干粉针制剂及其制备方法
US8975242B2 (en) 2005-03-14 2015-03-10 Wyeth Llc Tigecycline compositions and methods of preparation

Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1568999A (zh) * 2003-07-14 2005-01-26 南宁枫叶药业有限公司 稳定的医药用河豚毒素冷冻干燥制剂
CA2581083A1 (en) * 2004-09-22 2006-03-30 Helmut Henrich Buschmann Tetrodotoxin and its derivatives for the treatment of peripheral-nervously derived neuropathic pain
WO2007025212A2 (en) * 2005-08-25 2007-03-01 Wex Pharmaceuticals, Inc. Use of sodium channel blockers for the treatment of visceral pain or pain caused by cancer treatment
WO2007025213A2 (en) * 2005-08-25 2007-03-01 Wex Pharmaceuticals, Inc. Use of sodium channel blockers for the management of musculoskeletal pain
US8158152B2 (en) * 2005-11-18 2012-04-17 Scidose Llc Lyophilization process and products obtained thereby
EP2012774A1 (en) * 2006-03-27 2009-01-14 Wex Pharmaceuticals, Inc Use of sodium channel blockers for the treatment of neuropathic pain developing as a consequence of chemotherapy
CN101239061B (zh) * 2006-10-18 2011-05-11 江苏宏锦天药业有限公司 氨基全氢化喹唑啉类化合物清除生物体内瘦肉精的用途
WO2008088174A1 (en) * 2007-01-19 2008-07-24 Sang Gil Lee The composition comprising swellfish extracts using protease, and a manufacturing method thereof
CN101450056B (zh) * 2007-12-06 2013-04-24 江苏宏锦天药业有限公司 钠离子通道阻断剂用于治疗抗生素对生物耐药性的新用途
CN101554368B (zh) * 2008-04-07 2013-06-12 江苏宏锦天药业有限公司 一种室温稳定的注射用替曲朵辛组合物制剂
CN102020653A (zh) * 2009-09-22 2011-04-20 江苏宏锦天药业有限公司 氨基全氢化喹唑啉类化合物及其衍生物制备提高抗癌药药效的新应用
IN2013MU01127A (zh) 2013-03-26 2015-05-01 Astron Res Ltd
KR101398764B1 (ko) 2013-08-29 2014-05-27 강릉원주대학교산학협력단 입자의 이동에 의해 분석물질을 검출하는 장치 및 방법
CN111122752B (zh) * 2020-02-26 2022-06-10 浙江清华长三角研究院 一种河豚毒素成分分析标准物质的制备方法
CN112704665A (zh) * 2021-01-08 2021-04-27 江苏宏锦天药业有限公司 替曲朵辛口服制剂、替曲朵辛冻干片及其应用
CA3215362A1 (en) 2021-04-23 2022-10-27 Meng Zhou Tetrodotoxin liquid formulations
CN115475151A (zh) * 2022-08-14 2022-12-16 中国人民解放军海军军医大学 河豚毒素缓释微球及其制备方法和用途

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995024903A1 (en) * 1994-03-17 1995-09-21 Nanning Maple Leaf Pharmaceutical Co., Ltd. The use of amino hydrogenated quinazoline compounds and derivatives thereof for abstaining from drug dependence
CN1145225A (zh) * 1996-09-24 1997-03-19 王维国 用于戒毒、镇痛的药剂及其制法

Family Cites Families (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2912361A (en) * 1956-11-28 1959-11-10 Sterling Drug Inc Canine distemper vaccine and its preparation
JPS5967228A (ja) * 1982-10-07 1984-04-16 Green Cross Corp:The 寒冷不溶性グロブリンの凍結乾燥方法
US4816568A (en) * 1986-05-16 1989-03-28 International Minerals & Chemical Corp. Stabilization of growth hormones
AU2309692A (en) * 1991-07-03 1993-02-11 Cryolife, Inc. Method for stabilization of biomaterials
EP0857484A4 (en) 1995-09-13 2000-12-06 Nippon Shinyaku Co Ltd PGE1- FREEZING DRIED PREPARATION AND METHOD FOR THE PRODUCTION THEREOF
JP2937135B2 (ja) * 1995-09-13 1999-08-23 日本新薬株式会社 Ｐｇｅ１含有凍結乾燥製剤及び製法
US6030975A (en) * 1997-03-14 2000-02-29 Basf Aktiengesellschaft Carboxylic acid derivatives, their preparation and use in treating cancer
US6030974A (en) * 1997-04-02 2000-02-29 The Regents Of The University Of California Method of anesthesia
WO1998051290A2 (en) 1997-05-16 1998-11-19 Children's Medical Center Corporation Local anesthetic formulations comprising a site 1 sodium channel blocker combined with a second active agent
AU757002B2 (en) * 1998-03-03 2003-01-30 Eli Lilly And Company Pharmaceutical compositions containing the phospholipase inhibitor sodium ((3-(2-amino-1,2-dioxoethyl)-2-ethyl-1- phenylmethyl)-1H-indol-4-YL)oxy)acetate
US6306423B1 (en) * 2000-06-02 2001-10-23 Allergan Sales, Inc. Neurotoxin implant
CN1284536C (zh) * 2000-09-18 2006-11-15 威克斯医药有限公司 河豚毒素或蛤蚌毒素及其类似物在制备用于全身镇痛的镇痛药中的应用
CN1203857C (zh) 2000-09-18 2005-06-01 威克斯医药有限公司 局部麻醉与镇痛的新方法
CN1240702C (zh) 2000-09-18 2006-02-08 威克斯医药有限公司 高获得率提取替曲朵辛的方法
CN1187356C (zh) 2000-11-22 2005-02-02 南宁枫叶药业有限公司 高获得率提取替曲朵辛的系统
CN1187355C (zh) 2000-11-22 2005-02-02 南宁枫叶药业有限公司 精制高纯度替曲朵辛的方法
CN1236773C (zh) * 2000-11-22 2006-01-18 南宁枫叶药业有限公司 用于镇痛、麻醉或治疗药物依赖性的制剂
CN1269482C (zh) 2001-05-18 2006-08-16 威克斯医药有限公司 钠离子通道阻断剂和阿片类镇痛剂在制备用于对哺乳动物进行协同镇痛的药物中的应用
CN1203860C (zh) 2001-06-22 2005-06-01 威克斯医药有限公司 钠离子通道阻断剂和阿司匹林在制备用于对哺乳动物进行协同镇痛的药物中的应用
CN100360133C (zh) 2002-05-23 2008-01-09 潘心富 用于戒毒、镇痛的河豚毒素呼吸道给药制剂
CN1158285C (zh) * 2002-06-24 2004-07-21 厦门一元生物工程有限公司 河豚毒素高纯单体规模化制备方法
CN1568999A (zh) * 2003-07-14 2005-01-26 南宁枫叶药业有限公司 稳定的医药用河豚毒素冷冻干燥制剂
US20050197303A1 (en) 2003-10-31 2005-09-08 Bausch & Lomb Incorporated Combination of loteprednol etabonate and tobramycin for topical ophthalmic use
EP1799219A1 (en) 2004-09-21 2007-06-27 Laboratorios del Dr. Esteve S.A. Tetrodotoxin and its derivatives for the treatment of central-nervously derived neuropathic pain
CA2581083A1 (en) 2004-09-22 2006-03-30 Helmut Henrich Buschmann Tetrodotoxin and its derivatives for the treatment of peripheral-nervously derived neuropathic pain
WO2007025212A2 (en) 2005-08-25 2007-03-01 Wex Pharmaceuticals, Inc. Use of sodium channel blockers for the treatment of visceral pain or pain caused by cancer treatment
EP2012774A1 (en) 2006-03-27 2009-01-14 Wex Pharmaceuticals, Inc Use of sodium channel blockers for the treatment of neuropathic pain developing as a consequence of chemotherapy

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995024903A1 (en) * 1994-03-17 1995-09-21 Nanning Maple Leaf Pharmaceutical Co., Ltd. The use of amino hydrogenated quinazoline compounds and derivatives thereof for abstaining from drug dependence
CN1145225A (zh) * 1996-09-24 1997-03-19 王维国 用于戒毒、镇痛的药剂及其制法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP1645277A4 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8975242B2 (en) 2005-03-14 2015-03-10 Wyeth Llc Tigecycline compositions and methods of preparation
US9254328B2 (en) 2005-03-14 2016-02-09 Wyeth Llc Tigecycline compositions and methods of preparation
US9694078B2 (en) 2005-03-14 2017-07-04 Wyeth Llc Tigecycline compositions and methods of preparation
US10588975B2 (en) 2005-03-14 2020-03-17 Wyeth Llc Tigecycline compositions and methods of preparation
EP1858488B2 (en) † 2005-03-14 2022-07-20 Wyeth LLC Tigecycline compositions and methods of preparation
WO2010031346A1 (zh) * 2008-09-17 2010-03-25 厦门朝阳生物工程有限公司 河豚毒素冻干粉针制剂及其制备方法

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