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WO2010031346A1 - 河豚毒素冻干粉针制剂及其制备方法 - Google Patents

河豚毒素冻干粉针制剂及其制备方法 Download PDF

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Publication number
WO2010031346A1
WO2010031346A1 PCT/CN2009/074006 CN2009074006W WO2010031346A1 WO 2010031346 A1 WO2010031346 A1 WO 2010031346A1 CN 2009074006 W CN2009074006 W CN 2009074006W WO 2010031346 A1 WO2010031346 A1 WO 2010031346A1
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Prior art keywords
tetrodotoxin
freeze
per dose
dried powder
powder preparation
Prior art date
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Ceased
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PCT/CN2009/074006
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English (en)
French (fr)
Inventor
易瑞灶
陈晖�
洪碧红
谢荣伟
陈伟珠
洪专
徐淑贞
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Xiamen Zhaoyang Biological Engineering Co Ltd
Third Institute of Oceanography SOA
Original Assignee
Xiamen Zhaoyang Biological Engineering Co Ltd
Third Institute of Oceanography SOA
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Priority to JP2011527192A priority Critical patent/JP2012502932A/ja
Priority to CA2737463A priority patent/CA2737463C/en
Priority to US13/063,931 priority patent/US20110201627A1/en
Priority to MX2011002924A priority patent/MX2011002924A/es
Priority to EP09814066.8A priority patent/EP2397143A4/en
Priority to MX2011002851A priority patent/MX2011002851A/es
Application filed by Xiamen Zhaoyang Biological Engineering Co Ltd, Third Institute of Oceanography SOA filed Critical Xiamen Zhaoyang Biological Engineering Co Ltd
Priority to AU2009295083A priority patent/AU2009295083B2/en
Priority to RU2011109869/15A priority patent/RU2519654C2/ru
Publication of WO2010031346A1 publication Critical patent/WO2010031346A1/zh
Priority to ZA2011/02010A priority patent/ZA201102010B/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • 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
    • 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

Definitions

  • the invention relates to a tetrodotoxin (TTX) freeze-dried powder preparation and a preparation method thereof.
  • the present invention relates to a lyophilized powder preparation having a well-quantized tetrodotoxin as a main active ingredient and containing a suitable shaped skeleton excipient and a stabilizer.
  • the lyophilized powder preparation of the present invention can be used to block the dependence syndrome of opioids and cannabis drugs such as ice and heroin, and has a rapid onset and remarkable effect. Further, the lyophilized powder preparation of the present invention is stable, safe, and less irritating to the body. Background technique
  • Tetrodotoxin is a natural non-protein neurotoxin with the following chemical structure:
  • tetrodotoxin can be used as a detoxification agent to block dependence syndromes of opioids and cannabis drugs such as ice and heroin. It can control the onset of drug addiction and eliminate withdrawal response, and will not Generate dependencies.
  • Tetrodotoxin crystals are quite stable to heat and remain at 40 ° C for 6 months, and the quality of tetrodotoxin crystals remains essentially unchanged (see Table 1).
  • the aqueous solution of tetrodotoxin is very sensitive to temperature, is highly susceptible to degradation by temperature, and the higher the temperature, the faster the degradation.
  • the conventional Hessin injection, ⁇ after 30 days, its content decreased from 99.20% to 65.57%, a significant drop of 33.63% (see Table 2).
  • a stable tetrodotoxin freeze-dried formulation comprising a disaccharide such as lactose, sucrose, maltose and cellobiose and a glycan such as condensed glucose, dextran or a derivative thereof such as hydroxyethyl is disclosed in CN 1835754A A stabilizer for a starch or hydroxypropyl cyclodextrin in an amount of from 5 to 100 mg per dose.
  • the preparation further comprises a co-solvent selected from the group consisting of citric acid, tartaric acid, malic acid or lactobionic acid in an amount of from 0.00005 to 0.0005 mg per dose. It is pointed out in CN 1835754A that the obtained tetrodotoxin lyophilized preparation is unstable when using citrate or mannitol as an excipient, and the tetrodotoxin content gradually decreases during storage.
  • a co-solvent selected from the group consisting of citric acid, tartaric acid, malic acid or lactobionic acid in an amount of from 0.00005 to 0.0005 mg per dose.
  • the applicant conducted an in-depth study on the tetrodotoxin freeze-dried powder preparation and its manufacturing process.
  • the results showed that the freeze-dried powder injection skeleton excipient, tetrodotoxin stabilizer, cosolvent, pH range of the prescription solution, vacuum freeze-sublimation drying process, control of water content of powder injection preparation, etc. can significantly affect the powder injection preparation products.
  • Stability In particular, the wettability of the powder-formed skeleton carrier. The wettability of the excipients and the storage process over time prolonged the water content in the formulation, and the increase in water content will lead to a decrease in the content of the main drug puffer toxin in the preparation.
  • the present invention provides a tetrodotoxin freeze-dried powder preparation for safe injection, comprising tetrodotoxin, a solubilizing agent, a lyophilized excipient and a stabilizer, the tetrodotoxin having a purity of >96%, preferably 98% ⁇ 99.8%; the lyophilized excipient is sodium chloride or mannitol or a complex of the two; the stabilizer is dextran or trehalose or a combination of the two, and the cosolvent used It is citric acid.
  • the ratio of tetrodotoxin: excipient: stabilizer is preferably 1: 150-3000: 50-500 or 50-6000.
  • the tetrodotoxin content is from 0.1 to 20.0 ⁇ per dose, preferably from 0.5 to 20.0 ⁇ per dose, more preferably from 0.5 to 12.0 ⁇ per dose.
  • the content of the sodium chloride of the excipient is 1.0 to 30 mg per dose, preferably 5.0 to 30 mg per dose, more preferably 5.0 to 20 mg per dose.
  • the excipient mannitol is contained in an amount of 1.0 to 30 mg per dose, preferably 1.0 to 20 mg per dose, more preferably 3.0 to 10 mg per dose.
  • the stabilizer dextran is contained in an amount of from 0.5 to 5.0 mg per dose, preferably from 2.0 to 5.0 mg per dose, more preferably from 3.0 to 5.0 mg per dose.
  • the stabilizer trehalose content is
  • From 0.5 to 60 mg per dose preferably from 2.0 to 60 mg per dose, more preferably from 10 to 60 mg per dose.
  • the content of citric acid is from 0.001 to 0.080 mg per dose, preferably from 0.010 to 0.080 mg per dose, more preferably from 0.020 to 0.060 mg per dose.
  • the tetrodotoxin freeze-dried powder preparation of the present invention it is preferred to further contain a function regulating agent, and the function regulating agent is preferably lidocaine hydrochloride.
  • an inert gas such as high purity nitrogen or high purity carbon dioxide gas.
  • the tetrodotoxin freeze-dried powder preparation of the present invention is preferably administered in the form of administration by intramuscular or subcutaneous injection, and when dissolved in sterile water for injection, the amount of sterile water for injection is 0.5 to 2.0 ml per dose.
  • the present invention provides a method of preparing a tetrodotoxin freeze-dried powder preparation of the present invention, the method comprising the steps of:
  • a predetermined amount of tetrodotoxin is directly dissolved in a solution containing a cosolvent to adjust the pH to 3.0 to 6.0; preferably, the pH is 3.5 to 4.5, and the pyrogen is removed by filtration.
  • step (1) of the process of the invention filtration is preferably carried out by ultrafiltration.
  • the amount of the activated carbon is preferably 0.1 to 6.0 g / 100 ml.
  • it is filtered through a submicron microfiltration membrane or a charged microporous membrane of 0.05 ⁇ m to 0.20 ⁇ m.
  • Figure 1 shows the HPLC fluorescence detection pattern of tetrodotoxin injection at 40 ° C for 0 days.
  • Figure 2 shows the HPLC fluorescence detection pattern of tetrodotoxin injection at 40 ° C for 10 days.
  • Figure 3 is a HPLC fluorescence detection spectrum of a prescription A1 tetrodotoxin freeze-dried powder preparation at 40 ° C, 0 days.
  • Figure 4 is a HPLC fluorescence spectrum of the prescription A1 tetrodotoxin freeze-dried powder preparation at 40 ° C for 10 days.
  • Figure 5 is a HPLC fluorescence spectrum of the prescription B1 tetrodotoxin freeze-dried powder preparation at 40 ° C, 0 days.
  • Figure 6 is a HPLC fluorescence spectrum of the prescription B1 tetrodotoxin freeze-dried powder preparation at 40 ° C for 10 days.
  • Figure 7 is a HPLC fluorescence spectrogram of the prescription C1 tetrodotoxin freeze-dried powder preparation at 40 ° C, 0 days.
  • Figure 8 is a HPLC fluorescence spectrum of a prescription C1 tetrodotoxin freeze-dried powder preparation at 40 ° C for 10 days.
  • Figure 9 is a HPLC fluorescence spectrum of the prescription D5 tetrodotoxin freeze-dried powder preparation at 40 ° C, 0 days.
  • Figure 10 is a HPLC fluorescence detection spectrum of a prescription D5 tetrodotoxin freeze-dried powder preparation at 40 ° C for 10 days.
  • Figure 11 is a HPLC fluorescence spectrum of the prescription E5 tetrodotoxin freeze-dried powder preparation at 40 ° C, 0 days.
  • Figure 12 shows the HPLC fluorescence detection profile of the prescription E5 tetrodotoxin freeze-dried powder preparation at 40 ° C for 10 days.
  • Figure 13 is a HPLC fluorescence spectrum of the prescription F5 tetrodotoxin freeze-dried powder preparation at 40 °C, 0 days.
  • Figure 14 is a HPLC fluorescence detection profile of a prescription F5 tetrodotoxin freeze-dried powder preparation at 40 ° C for 10 days. Detailed description of the invention
  • the content of tetrodotoxin in the tetrodotoxin freeze-dried powder preparation of the present invention is measured by high performance liquid chromatography (Chinese Pharmacopoeia 2005 Edition, Part II, Appendix VII).
  • content In the chromatogram recorded under the measurement the retention time of the main peak of the test solution should be consistent with the retention time of the main peak of the reference solution.
  • the chromatographic conditions employed were as follows: octylsilane-bonded silica gel was used as a filler, and sodium octanesulfonate phosphate buffer was used as a mobile phase.
  • the flow rate is 0.3 ml/min, the excitation wavelength is 365 nm, the emission wavelength is 510 nm; the post-column derivatization agent is 4 mol/L sodium hydroxide solution; the derivatizing agent flow rate is 0.2-0.5 ml/min, and the post-column derivatization temperature is 100 °C ⁇ 140 ° C.
  • the number of theoretical plates is not less than 2000 based on the peak of tetrodotoxin.
  • the specific measurement method is as follows: Take the tetrodotoxin freeze-dried powder preparation of the present invention (containing tetrodotoxin 10 ⁇ , precise water addition 2.0 ml to dissolve, precision measurement 20 ⁇ l, injection under the conditions of the intended color language, recording fluorescence detection chromatogram; Take the appropriate amount of tetrodotoxin reference substance. Determine the tetrodotoxin content per dose/bottle according to the external standard method. When the tetrodotoxin content is 90% ⁇ 110% of the labeled amount, it is consistent with the freeze-dried powder needle. Medicinal requirements.
  • Determination of related substances Take the tetrodotoxin freeze-dried powder preparation of the present invention, add water to make a solution containing 20 g of tetrodotoxin per lml, as a test solution; accurately measure 1.0 ml, place in a 25 ml volumetric flask, dilute with water To the mark, shake well and use as a control solution. Precisely measure 20 ⁇ 1 of the test solution and the solution solution, respectively, and inject them into the liquid chromatograph, and record the fluorescence detection chromatogram to twice the retention time of the main component peak. When the sum of the impurity peak areas in the chromatogram of the test solution is not larger than the main peak area of the control solution, it meets the medicinal requirements.
  • the content of tetrodotoxin and related substances is detected by high performance liquid chromatography with fluorescence detection.
  • the high performance liquid chromatography (HPLC) fluorescence detection method has higher sensitivity and higher measurement accuracy, and thus can be better used for determining the stability of the tetrodotoxin preparation product.
  • the HPLC limit of HPLC is 8.14 ng
  • the HPLC method is 0.40 ng, indicating that the detection limit of fluorescence is 20 times lower than that of UV.
  • the limit of quantitation of the HPLC method was 20.26 ng, while the HPLC method was 0.81 ng, indicating that the limit of quantitation of the fluorescence method was 25 times lower than that of the UV method.
  • the common specification of the tetrodotoxin preparation product for injection is 10 g/bottle, and after sampling and dissolving, the injection concentration of the preparation product for detection is 5 g/m b 20 g/ml, when the injection amount is 20 ⁇ l.
  • the amount of the shield is only 1 - 4 ng, which is lower than the detection limit in the liquid chromatography UV detection method. If the sample contains 2% impurities, the impurity quality is only 2 - 8 Ng, also lower than the detection limit in the liquid chromatography UV detection method.
  • the detection limit of the liquid chromatography fluorescence detection method is 0.40 ng, which can fully meet the detection requirements.
  • the clinical dose of tetrodotoxin is in the microgram range, so excipient excipients need to be added as the forming skeleton of the freeze-dried powder preparation.
  • the screening of stabilizers is also crucial.
  • sodium chloride or / and mannitol were selected as excipients for the tetrodotoxin freeze-dried powder preparation, and dextran 20 or trehalose was selected as the stabilizer to obtain good results.
  • Formulations of representative tetrodotoxin freeze-dried powder formulations of the present invention comprising sodium chloride and/or mannitol as excipients and comprising dextran 20 or trehalose as stabilizers are summarized in Tables 3 and 4 below. Stability test results at 40 °C.
  • Table 4 Stability test results of different prescription tetrodotoxin freeze-dried powder formulations at 40 ° C
  • the mannitol product as an excipient has a good appearance and is weak in hygroscopicity.
  • Dextran has a significant protective effect on tetrodotoxin freeze-dried preparations, but it is highly hygroscopic.
  • the content of dextran 20 in the preparation of the present invention is
  • Tetrodotoxin is a non-protein marine neurotoxin with a relative molecular mass of 319.27, usually in the form of "amphoteric molecules", which are the functional groups necessary for its activity. It is insoluble in water or organic solvent. It is easily dehydrated and decomposed in the presence of strong acid and strong alkali. It is relatively stable in weakly acidic aqueous solution. Therefore, the lyophilized powder preparation prepared from tetrodotoxin should be selected from suitable acidic solvent as cosolvent. Applicants refer to the Chinese Pharmacopoeia 2005 Edition, the Handbook of Pharmaceutical Excipients, and the Chinese Pharmaceutical Excipients.
  • the standard grade weak acid substances for injections such as citric acid, acetic acid, ascorbic acid and sodium dihydrogen phosphate commonly used in injections.
  • An analysis selection was made. According to the principle of "Technical Guidelines for the Study of Chemical Pharmaceutical Preparations", acetic acid is a volatile acid, and there is a loss of volatilization during the freeze-drying process, which is not conducive to the pH value control after reconstitution of the powder injection preparation; ascorbic acid meets light and at normal temperature
  • the physicochemical properties are not stable enough; while sodium dihydrogen phosphate is an acid salt, the acidity is weak, the pH value is only 4.56 at 5% concentration, and the pH adjustment range is narrow, which is not suitable for use as an acidic solvent for tetrodotoxin freeze-dried powder needle; Non-volatile weak acid, there is a large space for pH adjustment.
  • citric acid was selected as a co-solvent for the tetrodotoxin freeze-dried powder needle of the present invention.
  • the amount of the co-solvent citric acid is preferably from 0.001 mg to 0.080 mg per dose.
  • the pH in the injection solution is one of the important factors closely related to the stability of the effective pharmaceutical ingredient.
  • the applicant prescribed a tetrodotoxin freeze-dried powder, adjusted the different pH values with 0.1% citric acid solution, and freeze-dried to make powder products. According to the quality inspection items, the appearance, water resolubility and pH value were Indicators such as stability and stability.
  • the main stability test The product is placed at a high temperature of 40 ° C, sampled at 0, 5, 10 days, respectively, using liquid chromatography fluorescence detection The measurement is performed. The content of tetrodotoxin and related substances were calculated by area normalization method. The experimental results are shown in Table 5 below. Table 5 Effect of pH value of powder injection preparation on stability of tetrodotoxin
  • the pH of the Hessian powder preparation solution has a significant influence on the stability of tetrodotoxin.
  • the tetrodotoxin freeze-dried powder preparation of the present invention preferably has a pH of 3.5 to 4.5 and an optimum pH of about 4.0. Effect of different water content on stability of tetrodotoxin freeze-dried powder products
  • the experiment shows that: when the water content of the freeze-dried powder product is more than 5%, the appearance of the product shrinks under the condition of 40 °C, and the stability is poor.
  • the water content of the tetrodotoxin freeze-dried powder preparation of the present invention is preferably controlled to be less than 3%. Freeze-drying process selection
  • the freeze-drying process of tetrodotoxin freeze-dried powder preparation mainly consists of three stages: pre-freezing, main drying and post-drying. According to the eutectic point of the prescription product and the use of the excipient and the stabilizer
  • the lyophilization process designed five freeze-drying conditions for testing (see Table 7). Through the comparative analysis of the test results, the control parameters of the optimum freeze-drying process of the product were determined (see Table 8). Selection of freeze-drying process for tetrodotoxin freeze-dried powder preparation
  • Example 1 Tetrodotoxin freeze-dried powder preparation containing sodium chloride as an excipient, dextran 20 as a stabilizer and containing citric acid as a solubilizing agent
  • a tetrodotoxin freeze-dried powder preparation having the following composition was prepared:
  • Preparation method Take a prescribed amount of Hesin, dissolve it with 10 ml of 0.1% citric acid solution, add a prescribed amount of sodium chloride, and add water for injection to about 250 ml. Adjust to the specified pH with 0.1% citric acid solution, filter the pyrogen to obtain the A component solution by ultrafiltration membrane; take the prescribed amount of dextran 20, add 200ml of water for injection and dissolve it, and adjust to the specified with 0.1% citric acid solution. The pH value is added to the weight-to-volume ratio of 0.1% to 1.0% of the activated carbon and stirred at 60 ° C for 30 minutes. The carbon is removed by filtration, the pyrogen is removed, and the mixture is cooled to room temperature to obtain a B component solution.
  • the function regulator lidocaine hydrochloride may optionally be contained in an amount of 5 mg/dose.
  • Tetrodotoxin freeze-dried powder preparation containing mannitol as an excipient, dextran 20 as a stabilizer and citric acid as a solubilizing agent
  • a tetrodotoxin freeze-dried powder preparation having the following composition was prepared:
  • Preparation method Take a prescribed amount of Hesin, dissolve it with 10 ml of 0.1% citric acid solution, and dilute it with water for injection to about 250 ml. Adjust to the specified pH with 0.1% citric acid solution, filter the pyrogen to obtain the A component solution by ultrafiltration membrane; take the prescribed amount of dextran 20 and mannitol, add 200ml of water for injection and dissolve it, adjust with 0.1% citric acid solution. To the specified pH, 0.2% by weight of activated carbon was added and stirred at 60 ° C for 30 minutes. The charcoal was removed by filtration, the pyrogen was removed, and the mixture was cooled to room temperature to obtain a B component solution.
  • the function regulator lidocaine hydrochloride may optionally be contained in an amount of 3.0 mg/dose.
  • Tetrodotoxin freeze-dried powder preparation containing sodium chloride as an excipient, trehalose as a stabilizer, and citric acid as a solubilizing agent
  • a tetrodotoxin freeze-dried powder preparation having the following composition was prepared:
  • Preparation method Take a prescribed amount of Hesin, dissolve it with 20 ml of 0.1% citric acid solution, and add a prescribed amount of sodium chloride and trehalose plus water for injection to dilute to about 450 ml. Adjust to the specified pH value with 0.1% citric acid solution, filter the pyrogen by ultrafiltration membrane, dilute to 500ml with water for injection, filter with 0.22 ⁇ microporous membrane, and check the pH value, clarity and content after sampling. Aseptic dispensing. After pre-freezing at -35 ° C for 2 to 6 hours, -10 ° C ⁇ 20 ° C main drying for 10 to 20 hours, 20 ° C ⁇ 50 ° C and drying for 6 to 10 hours, that is.
  • the function regulator lidocaine hydrochloride may optionally be contained in an amount of 3.0 mg/dose.
  • Example 4 Tetrodotoxin freeze-dried powder preparation containing mannitol as an excipient, trehalose as a stabilizer, and citric acid as a solubilizing agent
  • a tetrodotoxin freeze-dried powder preparation having the following composition was prepared:
  • Preparation method Take the prescribed amount of tetrodotoxin, add 20ml of 0.1% citric acid solution, add the prescription amount of trehalose, and add water for injection to about 300ml. Adjust to the specified pH with 0.1% citric acid solution, filter the pyrogen to obtain the A component solution; take the prescribed amount of mannitol, add 150ml of water for injection, and adjust with 0.1% citric acid solution. The specified pH value is added to the activated carbon at a weight ratio of 0.1% to 1.0% and stirred at 60 ° C for 30 minutes. The carbon is removed by filtration, the pyrogen is removed, and the mixture is cooled to room temperature to obtain a B component solution.
  • the functional modulator lidocaine hydrochloride may optionally be contained in an amount of 3.0 mg/dose.
  • Example 5 Tetrodotoxin freeze-dried powder preparation containing mannitol and sodium chloride as excipients, trehalose as a stabilizer, and citric acid as a solubilizing agent
  • a tetrodotoxin freeze-dried powder preparation having the following composition was prepared: Composition of tetrodotoxin mannitol sodium chloride trehalose before lyophilization
  • Prescription E6 20 5 9 40 4.5 Preparation method: Take the prescribed amount of tetrodotoxin, add 20ml of 0.1% citric acid solution, add the prescribed amount of sodium chloride and trehalose, and dilute with water for injection to about 300ml. Adjust to the specified pH with 0.1% citric acid solution, filter the pyrogen to obtain the A component solution; take the prescribed amount of mannitol, add 150ml of water for injection, and adjust to the specified with 0.1% citric acid solution. The pH value, the weight-to-volume ratio of 0.1% to 1.0% of activated carbon is stirred at 60 ° C for 30 minutes, the carbon is removed by filtration, the pyrogen is removed, and the mixture is cooled to room temperature to obtain a B component solution.
  • the functional modulator lidocaine hydrochloride may optionally be contained in an amount of 3.0 mg/dose.
  • Example 6 Tetrodotoxin freeze-dried powder preparation containing mannitol and sodium chloride as excipients, dextran 20 as a stabilizer and citric acid as a solubilizing agent
  • a tetrodotoxin freeze-dried powder preparation having the following composition was prepared: Composition of tetrodotoxin mannitol sodium chloride dextrose before lyophilization
  • Prescription F6 20 3 9 5 4.5 Preparation method: Take the prescribed amount of serotonin, add 20ml of 0.1% citric acid solution, add the prescription amount of mannitol, sodium chloride, and dilute with water for injection to about 300ml. Adjust to the specified pH with 0.1% citric acid solution, filter the pyrogen to obtain the A component solution; take the prescribed amount of dextran 20, add 150ml of water for injection, and adjust with 0.1% citric acid solution. The specified pH value, weight-to-volume ratio of 0.1% to 1.0% activated carbon and stirred at 60 ° C for 30 minutes, filtered to remove charcoal, in addition to pyrogen, cooled to room temperature to obtain a B component solution.
  • the functional modulator lidocaine hydrochloride may optionally be contained in an amount of 3.0 mg/dose.
  • the tetrodotoxin freeze-dried powder preparation prepared in the above examples was subjected to a high temperature stability test at 40 ° C for 10 days.
  • the specific operation of the test is as follows: Each prescription freeze-dried product was placed in a drug stability test chamber at a temperature of 40 ⁇ 2 ° C and a relative humidity of 75% ⁇ 5%. After 10 days, it was taken out and determined by high performance liquid chromatography with fluorescence. The purity of TTX indicates the formulation. The degree of stability.
  • the maximum amount is 2 ⁇ 3g, the minimum is 0.1g.
  • the drug abuse type is heroin, and the heroin is used together with heroin.
  • the drug-using method is hot, intramuscular or intravenous heroin.
  • the dosage of the preparation was 10ug-20ug / day, and the number of days used was 3 ⁇ 7 days.
  • the overall withdrawal symptoms of opioid drug dependent patients after discontinuation vary from person to person. According to the size of the patient's addiction and the severity of the withdrawal symptoms, the preparation of the present invention is administered at the appropriate time. The statistical results show that 95% of patients with withdrawal symptoms are eliminated within 0.5 ⁇ 3 hours after one dose of medication, and the patient remains awake and painless. After the first withdrawal symptoms are eliminated, it can last for 6 to 12 hours. When the patient has re-existing withdrawal symptoms, it is significantly less effective. For some patients with long-term drug use, patients with intravenous drug use and drug addiction can be given injection within 24 hours. The preparation of the invention is sub-. All patients were able to easily pass the most intense period of withdrawal symptoms without pain in waking state. After the withdrawal symptoms are eliminated, the patient's spirit is good, and the appetite and physical strength are quickly restored. Generally, no treatment is needed after 3 to 6 times of medication. There were no side symptoms and discomfort in all cases. Typical embodiment

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Description

河豚毒素冻干粉针制剂及其制备方法 技术领域
本发明涉及河豚毒素 (TTX)冻干粉针制剂及其制备方法。 具体地讲， 本发明涉及一种以准确定量的河豚毒素作为主要有效成分， 并含有适宜的 成型骨架赋形剂和稳定剂的冻干粉针制剂。 本发明的冻干粉针制剂可用于 阻断冰毒类、 海洛因等阿片类和大麻类毒品的依赖性戒断综合症并且起效 迅速、 效果显著。 此外， 本发明的冻干粉针制剂稳定、 安全并且对肌体刺 激性小。 背景技术
河豚毒素是一种天然的非蛋白质神经毒素， 其化学结构式如下:
Figure imgf000002_0001
化学名为： 八氢 -12- (羟曱基) -2-亚 > J^5，9:7，10a-二曱桥 -10aH-[l，3]二氧杂 环辛三烯并 [6，5-d]嘧啶 -4，7，10，11，12-五醇， 英文名称为 Tetrodotoxin, 简称 ττχ。
研究发现， 河豚毒素可以作为戒毒剂用于阻断冰毒类、 海洛因等阿片 类和大麻类毒品的依赖性戒断综合症，可以控制毒瘾发作并消除戒断反应， 并且在停用后不会产生依赖性。
河豚毒素晶体对热相当稳定， 在 40°C条件下保持 6个月， 河豚毒素晶 体的质量基本保持不变 (见表 1)。 但是河豚毒素的水溶液对温度则非常敏 感， 极易受温度的影响而发生降解， 且温度越高， 降解越快。 在 40°C条件 下， 常规的河 素注射、；^：置 30天后， 其含量由 99.20%降为 65.57%， 显著下降了 33.63% (见表 2)。 表 1 河豚毒素晶体 40°C高温下稳定性试验结果 *
Figure imgf000003_0001
表 2 河豚毒素酸性水溶液不同温度条件下稳定性试验结果
Figure imgf000003_0002
对于在水溶液中不稳定的药物而言， 为了获得稳定的可注射制剂， 将 其制成冻干制剂是本领域经常采用的一种常规方法。例如，在 CN 1835754A 中公开了一种稳定的河豚毒素冷冻干燥制剂，该制剂包含选自双糖如乳糖、 蔗糖、 麦芽糖和纤维二糖以及聚糖如缩合葡萄糖、 右旋糖酐或其衍生物如 羟乙基淀粉、 羟丙基环糊精的稳定剂， 其用量为每剂 5-100mg。 此外， 该 制剂中还包含选自杵檬酸、 酒石酸、 苹果酸或乳糖酸的助溶剂， 其用量为 每剂 0.00005-0.0005mg。 在 CN 1835754A中指出， 在用柠檬酸盐或甘露糖 醇作为赋形剂时， 所获得的河豚毒素冻干制剂是不稳定的， 河豚毒素的含 量在贮存过程中会逐渐下降。
仍需研制安全稳定、 质量可控、 可长期贮存的药用制剂产品用于河豚 毒素的临床使用。 发明内容
为了获得稳定的河豚毒素制剂， 申请人对河豚毒素冻干粉针制剂及其 制作工艺进行了深入的研究。 结果发现， 冻干粉针成型骨架赋形剂、 河豚 毒素稳定剂、 助溶剂、 处方溶液的 pH范围、 真空冷冻升华干燥工艺、 粉 针制剂含水量的控制等因素均可显著影响粉针制剂产品的稳定性。 特别是 粉针成型骨架载体的引湿性。 引湿性强的辅料、 贮存过程随时间的延长制 剂中的含水量呈緩慢上升趋势， 而含水量的增加， 将导致制剂中主药河豚 毒素的含量下降。
通过大量实验研究， 本发明人发现， 当选用等渗调节剂氯化钠或甘露 醇为冻干粉针成型骨架辅料，以右旋糖酐 20或海藻糖为河豚毒素主药的稳 定剂并且在冷冻干燥前调节 pH值至 3.5 - 4.5时， 可获得制品外观呈白色 疏松饼状物的河豚毒素冻干粉针制剂， 该制剂主药剂量准确， 外观优良， 质量稳定性好、 安全性高， 符合人体安全注射使用的要求。
因此， 本发明提供了一种符合人体安全注射使用的河豚毒素冻干粉针 制剂， 其中含有河豚毒素、 助溶剂、 冻干赋形剂和稳定剂， 所述河豚毒素 的纯度 >96%， 优选 98%~99.8%； 所述的冻干赋形剂为氯化钠或甘露醇或 二者的复合物； 所述的稳定剂为右旋糖酐或海藻糖或二者的复合物， 并且 所用的助溶剂为杵檬酸。
在本发明的河豚毒素冻干粉针制剂中， 河豚毒素： 赋形剂： 稳定剂的 比例优选为 1: 150-3000: 50-500或 50-6000。
在本发明的河豚毒素冻干粉针制剂中， 河豚毒素的含量为 0.1 至 20.0μ^每剂量， 优选 0.5至 20.0μ^每剂量， 更优选 0.5至 12.0μ^每剂量。
在本发明的河豚毒素冻干粉针制剂中， 赋形剂氯化钠的含量为 1.0~30mg/每剂量， 优选 5.0~30mg/每剂量， 更优选 5.0~20mg/每剂量。
在本发明的河豚毒素冻干粉针制剂中， 赋形剂甘露醇的含量为 1.0~30mg/每剂量， 优选 1.0~20mg/每剂量， 更优选 3.0~10mg/每剂量。
在本发明的河豚毒素冻干粉针制剂中， 稳定剂右旋糖酐的含量为 0.5~5.0mg/每剂量， 优选 2.0~5.0mg/每剂量， 更优选 3.0~5.0mg/每剂量。 在本发明的河豚毒素冻干粉针制剂中， 稳定剂海藻糖的含量为
0.5~60mg/每剂量， 优选 2.0~60mg/每剂量， 更优选 10~60mg/每剂量。
在本发明的河豚毒素冻干粉针制剂中 ， 柠檬酸的含量为 0.001~0.080mg/每剂量，优选 0.010-0.080mg/每剂量，更优选 0.020-0.060mg/ 每剂量。
在本发明的河豚毒素冻干粉针制剂中， 优选还包含功能调节剂， 并且 所述的功能调节剂优选为盐酸利多卡因。
在本发明的河豚毒素冻干粉针制剂中， 优选充入惰性气体如高纯氮气 或高纯二氧化碳气体。
本发明的河豚毒素冻干粉针制剂优选为供肌肉或皮下注射给药的形 式， 并且在用无菌注射用水溶解时， 无菌注射用水的用量为 0.5~2.0ml/每 剂量。
此外， 本发明还提供了制备本发明的河豚毒素冻干粉针制剂的制备方 法， 该方法包括如下步骤：
(1)将预定量的河豚毒素直接溶入包含助溶剂的溶液中， 调节 pH值为 3.0-6.0; 优选 pH3.5-4.5， 过滤除热原。
(2)将冻干赋形剂和稳定剂以及任选的功能调节剂直接溶入无菌注射 用水中， 加活性炭搅拌 30分钟后， 过滤除热原。
(3)将 (1)和 (2)中获得的溶液合并混合均匀，过滤除菌，定量灌装于西林 瓶中， 真空冷冻干燥， 充入惰性气体， 压塞后扎盖， 得冻干粉针制剂产品。
在本发明方法的步骤 (1)中， 优选通过超滤进行过滤。
在本发明方法的步骤 (2)中， 活性炭的用量优选为 0.1~6.0g/100ml。 在本发明方法的步骤 (3)中，经 0.05μιη~0.20μιη亚微米级微孔滤膜或荷 电微孔滤膜过滤。 附图说明
图 1为河豚毒素注射液 40°C， 0天时的 HPLC荧光检测图语。
图 2为河豚毒素注射液 40°C， 10天时的 HPLC荧光检测图语。 图 3为处方 A1河豚毒素冻干粉针制剂 40°C， 0天时的 HPLC荧光检 测图谱。
图 4为处方 A1河豚毒素冻干粉针制剂 40°C， 10天时的 HPLC荧光检 测图谱。
图 5为处方 B1河豚毒素冻干粉针制剂 40°C， 0天时的 HPLC荧光检 测图谱。
图 6为处方 B1河豚毒素冻干粉针制剂 40°C， 10天时的 HPLC荧光检 测图谱。
图 7为处方 C1河豚毒素冻干粉针制剂 40°C， 0天时的 HPLC荧光检 测图谱。
图 8为处方 C1河豚毒素冻干粉针制剂 40°C， 10天时的 HPLC荧光检 测图谱。
图 9为处方 D5河豚毒素冻干粉针制剂 40°C， 0天时的 HPLC荧光检 测图谱。
图 10为处方 D5河豚毒素冻干粉针制剂 40°C， 10天时的 HPLC荧光 检测图谱。
图 11为处方 E5河豚毒素冻干粉针制剂 40°C， 0天时的 HPLC荧光检 测图谱。
图 12为处方 E5河豚毒素冻干粉针制剂 40°C， 10天时的 HPLC荧光 检测图谱。
图 13为处方 F5河豚毒素冻干粉针制剂 40°C， 0天时的 HPLC荧光检 测图谱。
图 14为处方 F5河豚毒素冻干粉针制剂 40°C， 10天时的 HPLC荧光 检测图谱。 发明详述
在本说明书中， 对本发明的河豚毒素冻干粉针制剂中河豚毒素含量的 测定采用高效液相色谱法 (中国药典 2005版二部附录 VII)来进行。 在含量 测定项下记录的色谱图中， 供试品溶液主峰的保留时间应与对照品溶液主 峰的保留时间一致。
所采用的色谱条件如下： 用辛烷基硅烷键合硅胶为填充剂， 以辛烷磺 酸钠的磷酸盐緩冲液为流动相。 流速为 0.3ml/分钟， 激发波长为 365nm， 发射波长为 510nm; 柱后衍生剂为 4mol/L氢氧化钠溶液； 衍生剂流速为 0.2-0.5 ml/分钟， 柱后衍生温度为 100°C~140°C。 理论塔板数按河豚毒素峰 计算不低于 2000。
具体测定方法如下： 取本发明的河豚毒素冻干粉针制剂（含河豚毒素 10μ^， 精密加水 2.0ml使溶解， 精密量取 20μ1， 于拟定色语条件下进样， 记录荧光检测色谱图； 另取河豚毒素对照品适量。 同法测定。 按外标法以 峰面积计算每剂 /瓶的河豚毒素含量。 当河豚毒素的含量为标示量的 90%~110%时， 符合冻干粉针药用要求。
有关物质的测定：取本发明的河豚毒素冻干粉针制剂，加水制成每 lml 含河豚毒素 20 g的溶液， 作为供试品溶液； 精密量取 1.0ml, 置 25ml量 瓶中， 加水稀释至刻度， 摇匀， 作为对照溶液。 精密量取供试品溶液与对 照品溶液各 20μ1， 分别注入液相色语仪， 记录荧光检测色谱图至主成份峰 保留时间的 2倍。 当供试品溶液的色谱图中各杂质峰面积之和不大于对照 溶液主峰面积时， 符合药用要求。
在本发明中， 采用高效液相色谱荧光检测法对河豚毒素以及相关物质 的含量进行检测。 与常规的高效液相色谱紫外检测法相比， 高效液相色谱 荧光检测法的灵敏度更高， 测定准确度高， 因而可以更好地用于测定河豚 毒素制剂产品的稳定性。 例如， 对于河豚毒素制剂而言， 高效液相色谱紫 外检测法的最^ r出限为 8.14ng， 而高效液相色谱荧光法为 0.40ng， 表明 荧光法检测限低于紫外法 20倍。 此外， 对于河豚毒素制剂而言， 高效液相 色语紫外法的定量限为 20.26 ng， 而高效液相色谱荧光法为 0.81ng， 表明 荧光法的定量限低于紫外法 25倍。
具体而言， 注射用河豚毒素制剂产品的常用规格为 10 g/瓶， 取样溶 解后，用以检测的制剂产品进样浓度为 5 g/m卜 20 g/ml，当进样量为 20μ1 时， 若样品中含有 1%的杂质， 杂盾量仅 1 - 4 ng， 低于液相色谱紫外检测 法中的检测限； 若样品中含有 2%的杂质时， 杂质量仅为 2 - 8 ng， 也低于 液相色谱紫外检测法中的检测限。 可见， 用液相色谱紫外检测法检测制剂 中的有关杂质或含量时， 对于产品中低于 5%杂质， 可能因检测限无法满 足要求而无法检出， 因低于定量限而导致测定误差。 相反， 液相色谱荧光 检测法的检测限为 0.40ng， 完全能够达到检测要求。
采用以上测定条件， 申请人对影响河豚毒素冻干粉针制剂稳定性的各 种因素进行了研究。 河豚毒素冻干粉针赋形剂与稳定剂的筛选
河豚毒素的临床使用剂量为微克级， 因此需添加赋形剂辅料作为冻干 粉针制剂的成型骨架。 同时基于河豚毒素粉针制剂稳定性的需要， 稳定剂 的筛选也至关重要。 通过大量实验， 申请人发现， 选用氯化钠或 /和甘露醇 作为河豚毒素冻干粉针制剂赋形剂辅料，选择右旋糖酐 20或海藻糖为稳定 剂可以获得良好的结果。在下表 3和表 4中概述了包含氯化钠和 /或甘露醇 作为赋形剂并且包含右旋糖酐 20或海藻糖作为稳定剂的本发明代表性的 河豚毒素冻干粉针制剂的处方及其在 40 °C下的稳定性试验结果。
Figure imgf000008_0001
表 4: 不同处方河豚毒素冻干粉针制剂 40°C下稳定性试验结果
0天 5天 10天 处方 外观 含量 有关 含量 有关 含量 有关
(%) 物质 (%) 物质 (%) 物质
〈对照 〈对照 〈对照 饱满， 洁白， 呈 溶液 溶液 溶液
1 100 99.30 98.70
饼状物 主峰 主峰 主峰 面积 面积 面积
〈对照 >对照 >对照 白色饼状物，
溶液 溶液 溶液
2 40°C下 10天未 100 97.84 97.03
主峰 主峰 主峰 见明显变化
面积 面积 面积
〈对照 〈对照 〈对照 白色饼状物，
溶液 溶液 溶液
3 40°C下 10天未 100 99.06 98.41
主峰 主峰 主峰 见明显变化
面积 面积 面积
〈对照 >对照 >对照 白色饼状物，
溶液 溶液 溶液
4 40°C下 10天未 100 95.56 92.26
主峰 主峰 主峰 见明显变化
面积 面积 面积
〈对照 〈对照 〈对照 白色饼状物，
溶液 溶液 溶液
5 40°C下 10天未 100 98.96 98.59
主峰 主峰 主峰 见明显变化
面积 面积 面积
〈对照 〈对照 〈对照 白色饼状物，
溶液 溶液 溶液
6 40°C下 10天未 100 99.37 98.71
主峰 主峰 主峰 见明显变化
面积 面积 面积 其中作为赋形剂的甘露醇产品外观甚佳且吸湿性弱。右旋糖酐对河豚 毒素冻干制剂有显著的保护作用， 但其吸湿性强。 为控制冻干粉针制品的 含水量并且形成饱满的冻干成型骨架，本发明制剂中右旋糖酐 20的含量为
4mg/剂， 甘露醇的含量为 6mg/剂。 另一方面， 当以等渗调节剂氯化钠为赋 形剂成型骨架辅料， 以右旋糖酐 20或海藻糖为稳定剂组成处方时，制成品 含水量低、 外观呈饱满饼状物。 在实际应用中加水后迅速复溶。 40°C高温 下， 10天稳定性试验符合药用要求。 助溶剂与处方 pH值的选择
河豚毒素是一种相对分子质量为 319.27的非蛋白海洋神经毒素， 通常 以"两性分子"的形式存在， 结构中的胍基是其活性所必需的功能基。 不溶 于水或有机溶剂， 遇强酸和强碱时易脱水分解， 在弱酸性水溶液中相对稳 定， 因此河豚毒素制成的冻干粉针制剂应选择适宜的酸性溶媒为助溶剂。 申请人参考《中国药典 2005版》、 《药用辅料手册》和《中国药用辅料大 全》等资料， 对注射剂中常用的柠檬酸、 醋酸、 抗坏血酸和磷酸二氢钠等 注射剂标准级弱酸性物质进行了分析选择。 根据《化学药物制剂研究技术 指导原则》优先选用原则， 醋酸为挥发性酸， 在冷冻干燥过程中存在挥发 损失， 不利于粉针制剂加水复溶后 pH值的控制； 抗坏血酸遇光和在常温 下理化性质不够稳定； 而磷酸二氢钠为酸式盐， 酸性弱， 5%浓度下 pH值 仅为 4.56， pH调节范围窄， 不适于用作河豚毒素冻干粉针的酸性溶媒； 柠 檬酸为非挥发性弱酸， pH调节范围有较大的空间。 因此， 经分析和比较， 选择柠檬酸为本发明的河豚毒素冻干粉针的助溶剂。 在本发明的冻干粉针 制剂中， 助溶剂杵檬酸的用量优选为每剂量 O.OOlmg至 0.080mg。
注射剂溶液中的 pH值是与有效药用成分稳定性密切相关的重要因素 之一。本申请人以设定的河豚毒素冻干粉针处方，用 0.1%柠檬酸溶液调节 不同的 pH值， 经冷冻干燥制成粉针制品， 按照质量检查项目对其外观、 加水复溶性及 pH值、 稳定性等指标进行考察。 主要的稳定性试验： 将制 品置于 40°C高温下， 分别于 0、 5、 10天取样检查， 利用液相色谱荧光检 测法进行测定。 以面积归一化法计算河豚毒素的含量和有关物质。 实验结 果见下表 5。 表 5粉针制剂 pH值对河豚毒素稳定性的影响
Figure imgf000011_0001
从上述实验结果可以看出， 河 素粉针制剂溶液中的 pH对河豚毒 素的稳定性有显著影响。 在 40°C条件下， pH值大于 5时， 保持 10天， 河 豚毒素降解产生的有关物质大于对照溶液主峰面积， 并出现新的杂质峰。 经反复试验， 申请人发现本发明的河豚毒素冻干粉针制剂优选的 pH 值为 3.5-4.5， 最佳 pH值为 4.0左右。 河豚毒素冻干粉针制品不同含水量对稳定性的影响
申请人还对本发明的河豚毒素冻干粉针制品中不同含水量对稳定性的 影响进行了研究。 结果见表 6。
表 6: 不同含水量的河豚毒素冻干粉针制品 40°C下的稳定性试验结果
Figure imgf000012_0001
实验表明： 冻干粉针制品含水量大于 5%时， 制品外观在 40°C条件下 出现萎缩， 稳定性较差。 冻干制品的含水量越低， 越有利于制品中河豚毒 素的稳定。 本发明的河豚毒素冻干粉针制剂的含水量优选控制为小于 3%。 冻干工艺的选择
河豚毒素冻干粉针制剂的冷冻干燥工艺主要由预冻、 主干燥和后干燥 等三个阶段组成。 根据处方制品的共晶点和赋形剂与稳定剂筛选时采用的 冻干工艺设计了五种冻干条件方案进行试验 (参见表 7)。通过试验结果的比 较分析， 确定本制品的最佳冷冻干燥工艺的控制参数 (参见表 8)。 河豚毒素冻干粉针制剂冷冻干燥工艺的选择
Figure imgf000013_0001
由方案 1的试验发现， 制品经预冻于 -10°C低温真空干燥 15小时后， 干燥温度直接升到 40°C， 制品中河豚毒素含量呈显著下降。 表明冷冻干燥 工艺的设计对河豚毒素冻干粉针制品的质量有显著影响。 方案 4通过优化 干燥曲线， 主干燥阶段采用分阶段梯度升温至后干燥阶段采用 40°C干燥， 所得制品含水量低。 冻干工艺过程对制品中的河豚毒素含量无影响。 因此， 选择方案 4为本制品冷冻干燥的优选冻干工艺。 具体实施方案
实施例 1: 包含氯化钠作为赋形剂、 右旋糖酐 20作为稳定剂并且包含杵檬 酸作为助溶剂的河豚毒素冻干粉针制剂
制备具有如下组成的河豚毒素冻干粉针制剂：
Figure imgf000014_0001
制备方法： 取处方量的河 素， 用 10ml 0.1%柠檬酸溶液溶解后， 加入处方量的氯化钠， 加注射用水稀释至约 250ml。 用 0.1%柠檬酸溶液调 节至指定的 pH值， 超滤膜过滤除热原得 A组分溶液； 另取处方量的右旋 糖酐 20， 加 200ml注射用水溶解， 用 0.1%柠檬酸溶液调节至指定的 pH 值， 加入重量体积比 0.1%~1.0%活性炭在 60°C保温搅拌 30分钟， 过滤除 炭， 除热原， 冷却至室温得 B组分溶液。 将上述 A、 B两种溶液混句， 用 注射用水定容至 500ml， 用 0.22μιη微孔滤膜过滤， 取样检测 pH值、 澄明 度、 含量等合格后， 无菌分装。 -35°C预冻 2~6小时后， -10°C~20°C主干燥 10-20小时， 20°C~50°C后干燥 6~10小时，即得。产品外观为白色饼状物。 在上述河豚毒素冻干粉针制剂中， 还可任选地包含功能调节剂盐酸利 多卡因， 其用量可为 5mg/剂。 实施例 2: 包含甘露醇作为赋形剂、 右旋糖酐 20作为稳定剂并且包含杵檬 酸作为助溶剂的河豚毒素冻干粉针制剂
制备具有如下组成的河豚毒素冻干粉针制剂：
Figure imgf000015_0001
制备方法： 取处方量的河 素， 用 10ml 0.1%柠檬酸溶液溶解后， 加注射用水稀释至约 250ml。 用 0.1%柠檬酸溶液调节至指定的 pH值， 超 滤膜过滤除热原得 A组分溶液； 另取处方量的右旋糖酐 20和甘露醇， 加 200ml注射用水溶解， 用 0.1%柠檬酸溶液调节至指定的 pH值， 加入重量 体积比 0.2%的活性炭并在 60°C保温搅拌 30分钟， 过滤除炭， 除热原， 冷 却至室温得 B组分溶液。 将上述 A、 B两种溶液混句， 用注射用水定容至 500ml, 用 0.22μιη微孔滤膜过滤， 取样检测 pH值、 澄明度、 含量等合格 后， 无菌分装。 -35°C预冻 2~6 小时后， -10°C~20°C主干燥 10~20 小时， 20°C~50°C后干燥 6~10小时， 即得。 产品外观为白色饼状物。 在上述河豚毒素冻干粉针制剂中， 还可任选地包含功能调节剂盐酸利 多卡因， 其用量可为 3.0mg/剂。 实施例 3: 包含氯化钠作为赋形剂、 海藻糖作为稳定剂并且包含杵檬酸作 为助溶剂的河豚毒素冻干粉针制剂
制备具有如下组成的河豚毒素冻干粉针制剂：
Figure imgf000016_0001
制备方法： 取处方量的河 素， 用 20ml 0.1%柠檬酸溶液溶解后， 加入处方量的氯化钠和海藻糖加注射用水稀释至约 450ml。用 0.1%柠檬酸 溶液调节至指定的 pH值， 超滤膜过滤除热原， 注射用水定容至 500ml， 用 0.22μιη微孔滤膜过滤， 取样检测 pH值、 澄明度、 含量等合格后， 无菌 分装。 -35°C预冻 2~6小时后， -10°C~20°C主干燥 10~20小时， 20°C~50°C 后干燥 6~10小时， 即得。 产品外观为白色饼状物。 在上述河豚毒素冻干粉针制剂中， 还可任选地包含功能调节剂盐酸利 多卡因， 其用量可为 3.0mg/剂。 实施例 4: 包含甘露醇作为赋形剂、 海藻糖作为稳定剂并且包含杵檬酸作 为助溶剂的河豚毒素冻干粉针制剂
制备具有如下组成的河豚毒素冻干粉针制剂：
Figure imgf000017_0001
制备方法： 取处方量的河豚毒素， 加 20ml 0.1%柠檬酸溶液溶解后， 加入处方量的海藻糖， 加注射用水稀释至约 300ml。 用 0.1%杵檬酸溶液调 节至指定的 pH值， 超滤膜过滤除热原得 A组分溶液； 另取处方量的甘露 醇， 加 150ml注射用水溶解， 用 0.1%杵檬酸溶液调节至指定的 pH值， 加 入重量体积比 0.1%~1.0%活性炭并在 60°C保温搅拌 30分钟， 过滤除炭， 除热原， 冷却至室温得 B组分溶液。 将上述 A、 B两种溶液混匀， 用注射 用水定容至 500ml， 用 0.22μιη微孔滤膜过滤， 取样检测 pH值、 澄明度、 含量等合格后，无菌分装。 -35°C预冻 2~6小时后， -10°C~20°C主干燥 10~20 小时， 20°C~50°C后干燥 6~10小时， 即得。 产品外观为白色饼状物。
在上述河豚毒素冻干粉针制剂中， 还可任选地包含功能调节剂盐酸利 多卡因， 其用量可为 3.0mg/剂。
实施例 5: 包含甘露醇和氯化钠作为赋形剂、 海藻糖作为稳定剂并且包含 柠檬酸作为助溶剂的河豚毒素冻干粉针制剂
制备具有如下组成的河豚毒素冻干粉针制剂： 组成 河豚毒素 甘露醇 氯化钠 海藻糖 在冷冻干燥前用
(纯度 >99%) (mg/剂） (mg/剂） (mg/剂） 柠檬酸调节溶液
(μ§/剂） pH值至 处方 El 3 5 9 5 3.5
处方 E2 5 5 9 10 4.0
处方 E3 8 5 9 15 3.8
处方 E4 15 5 9 30 4.0
处方 E5 10 5 9 20 4.0
处方 E6 20 5 9 40 4.5 制备方法： 取处方量的河豚毒素， 加 20ml 0.1%柠檬酸溶液溶解后， 加入处方量的氯化钠和海藻糖， 加注射用水稀释至约 300ml。 用 0.1%柠檬 酸溶液调节至指定的 pH值， 超滤膜过滤除热原得 A组分溶液； 另取处方 量的甘露醇， 加 150ml注射用水溶解， 用 0.1%柠檬酸溶液调节至指定的 pH值， 重量体积比 0.1%~1.0%活性炭并在 60°C保温搅拌 30分钟， 过滤 除炭， 除热原， 冷却至室温得 B组分溶液。 将上述 A、 B两种溶液混匀， 用注射用水定容至 500ml， 用 0.22μιη微孔滤膜过滤， 取样检测 pH值、 澄 明度、 含量等合格后， 无菌分装。 -35°C预冻 2~6小时后， -10°C~20°C主干 燥 10~20小时， 20°C~50°C后干燥 6~10小时， 即得。 产品外观为白色饼状 物。
在上述河豚毒素冻干粉针制剂中， 还可任选地包含功能调节剂盐酸利 多卡因， 其用量可为 3.0mg/剂。 实施例 6: 包含甘露醇和氯化钠作为赋形剂、 右旋糖酐 20作为稳定剂并且 包含柠檬酸作为助溶剂的河豚毒素冻干粉针制剂
制备具有如下组成的河豚毒素冻干粉针制剂： 组成 河豚毒素 甘露醇 氯化钠 右旋糖 在冷冻干燥前用
(纯度 >99%) (mg/剂） (mg/剂） 酐 20 柠檬酸调节溶液
(μ§/剂） (mg/剂） pH值至
处方 Fl 3 3 9 4 3.5
处方 F2 5 3 9 4 3.5
处方 F3 10 3 9 4 4.0
处方 F4 15 3 9 5 4.2
处方 F5 10 3 9 4.5 4.0
处方 F6 20 3 9 5 4.5 制备方法： 取处方量的河 素， 加 20ml 0.1%柠檬酸溶液溶解后， 加入处方量的甘露醇、 氯化钠， 加注射用水稀释至约 300ml。 用 0.1%杵檬 酸溶液调节至指定的 pH值， 超滤膜过滤除热原得 A组分溶液； 另取处方 量的右旋糖酐 20， 加 150ml注射用水溶解， 用 0.1%杵檬酸溶液调节至指 定的 pH值， 重量体积比 0.1%~1.0%活性炭并在 60°C保温搅拌 30分钟， 过滤除炭， 除热原， 冷却至室温得 B组分溶液。 将上述 A、 B两种溶液混 匀，用注射用水定容至 500ml，用 0.22μιη微孔滤膜过滤，取样检测 pH值、 澄明度、 含量等合格后， 无菌分装。 -35°C预冻 2~6小时后， -10°C~20°C主 干燥 10~20小时， 20°C~50°C后干燥 6~10小时， 即得。 产品外观为白色饼 状物。
在上述河豚毒素冻干粉针制剂中， 还可任选地包含功能调节剂盐酸利 多卡因， 其用量可为 3.0mg/剂。
实施例 7: 稳定性试验
将以上实施例中制得的河豚毒素冻干粉针制剂进行 40°C高温 10天稳 定性试验。 试验的具体操作如下： 各处方冻干制品置温度 40 ± 2°C，相对湿度 75% ± 5%的药品稳定性实 验箱中， 10天后取出， 以高效液相色谱荧光法进行测定， 以 TTX的纯度 变化表示制剂的稳定程度。
在上述稳定性试验中， 本发明的河豚毒素冻干粉针制剂均表现出良好 的稳定性。 优选处方具体试验结果如下： 表 9: 本发明优选处方的河豚毒素冻干粉针制剂 40°C下 10天的稳定性试验 结果
Figure imgf000020_0001
应用实施例
应用本发明的制剂实施脱瘾治疗的吸毒患者共 80例， 其中男 49例， 女 31例， 年龄最大者 41岁， 最小者 18岁， 吸毒历史最长 13年， 最短 2 年， 每日吸毒量最大 2~3g， 最小 0.1g， 毒品滥用种类为海洛因、 与海洛因 并用镇静安眠药， 吸毒方式为烫吸、 肌肉注射或静脉注射海洛因。 80例患 者均符合 DSM - III - R精神活性物质所致的精神障碍——阿片类依赖的 诊断标准， 尿液吗啡类物质检测均呈阳性， 不伴有其它严重躯体疾病， 治 疗前均选药物依赖戒断症状诊为重度的吸毒患者， 本制剂用量 10ug-20ug / 天， 使用天数： 3~7天。
鸦片类毒品依赖患者停药后产生的综合戒断症状因人而异表现不同。 根据患者毒瘾的大小、 戒断症状发作程度的轻重， 分别适时给予注射本发 明制剂。 统计结果表明， 在一次用药后 0.5~3小时内， 95%的患者戒断症 状得到消除， 患者始终保持清醒状态且无痛苦。 首次戒断症状消除后即可 持续 6~12小时， 患者再次出现戒断症状时较前明显减轻，对部分吸毒时间 长，以静脉注射毒品且毒瘾大的患者在 24小时内可给予注射 2次本发明制 剂。全部患者均能在清醒状态下无痛苦地轻松度过最激烈的戒断症状时期。 戒断症状消除后患者精神佳， 食欲、 体力随之快速恢复。 一般经 3~6次用 药疗程后不再需要治疗。所有病例用药过程均未出现任何副症状和不适感。 典型实施例
患者 E.S, 男， 32岁， 吸毒时间 10年， 平均曰用量 1.0克， 毒品种类 为海洛因， 使用毒品方式为肌肉注射或静脉注射。 患者停用毒品， 自愿接 受戒毒治疗， 同日 19: 00时出现明显戒断症状， 21: 00时症状加重， 患 者体征： 流涕、 流泪、 呵欠、 骨肌疼痛、 寒颤、 腹痛、 腹泻、 恶心、 呕吐、 皮肤呈鸡皮疙瘩、汗毛竖立、躯体呈倦曲姿势等，戒断症状达到" + +，，~ " + + + "， 立即给予本制剂肌肉注射， 0.5小时后戒断症状明显得到控制， 2.5 小时后安静入睡， 无任何痛苦， 10小时后患者又出现戒断症状， 但戒断症 状程度较前明显减轻， 次日 9: 30, 再次注射本制剂， 注射后患者自我感 觉良好， 无明显不适。 间隔约 12小时后患者再次出现戒断症状并伴腹泻， 21: 00再次给予注射本制剂， 患者感觉良好， 一夜平静， 以后每日施以本 制剂注射， 至患者自觉明显好转， 腹泻彻底消失， 食欲恢复， 开始排正常 便， 至第 5日未出现戒断症状， 患者无其它不适。 治疗过程共 5天， 共用 本制剂 6次。

Claims

权 利 要 求
1、 一种河豚毒素冻干粉针制剂， 其中含有河豚毒素、 助溶剂、 冻干赋 形剂和稳定剂， 所述河豚毒素的纯度 >96%， 优选 98%~99.8%； 所述的冻 干赋形剂为氯化钠或甘露醇或二者的复合物； 所述的稳定剂为右旋糖酐或 海藻糖或二者的复合物， 并且所述的助溶剂为杵檬酸， 所述的制剂干燥方 法为真空冷冻升华干燥。
2、 权利要求 1所述的河豚毒素冻干粉针制剂， 其中， 河豚毒素： 赋形 剂： 稳定剂的比例为 1: 150-3000: 50-500或 50-6000。
3、权利要求 1所述的河豚毒素冻干粉针制剂，其中河豚毒素的含量为 0.1至 20.0μ^每剂量， 优选 0.5至 20.0μ^每剂量， 更优选 0.5至 12.0 g/每 剂量。
4、权利要求 1所述的河豚毒素冻干粉针制剂，其中赋形剂氯化钠的含 量为 1.0~30mg/每剂量， 优选 5.0-30mg/每剂量， 更优选 5.0-20mg/每剂量。
5、权利要求 1所述的河豚毒素冻干粉针制剂，其中赋形剂甘露醇的含 量为 1.0~30mg/每剂量， 优选 1.0~20mg/每剂量， 更优选 3.0-10mg/每剂量。
6、权利要求 1所述的河豚毒素冻干粉针制剂，其中稳定剂右旋糖酐的 含量为 0.5~5.0mg/每剂量， 优选 2.0~5.0mg/每剂量， 更优选 3.0~5.0mg/每 剂量。
7、权利要求 1所述的河豚毒素冻干粉针制剂，其中稳定剂海藻糖的含 量为 0.5~60mg/每剂量， 优选 2~60mg/每剂量， 更优选 10~60mg/每剂量。
8、 权利要求 1 所述的河豚毒素冻干粉针制剂， 其中杵檬酸的含量为 0.001~0.080mg/每剂量， 优选 0.010~0.080mg/每剂量， 更优选 0. 020~0.060mg/每剂量。
9、权利要求 1-8中任意一项所述的河豚毒素冻干粉针制剂， 其中还包 含功能调节剂盐酸利多卡因。
10、 权利要求 1-9中任意一项所述的河豚毒素冻干粉针制剂， 其中充 入了惰性气体如高纯氮气或高纯二氧化碳气体。
11、权利要求 1-10中任意一项所述的河豚毒素冻干粉针制剂， 其为供 肌肉或皮下注射给药的形式。
12、制备权利要求 1-11中任意一项所述的河豚毒素冻干粉针制剂的方 法， 该方法包括如下步骤：
(1)将预定量的河豚毒素以及任选的功能调节剂直接溶入包含助溶剂 的溶液中， 调节 pH值为 3.0~6.0； 优选 3.5-4.5， 过滤除热原。
(2)将冻干赋形剂和稳定剂直接溶入无菌注射用水中，加活性炭搅拌 30 分钟后， 过滤除热原。
(3)将 (1)和 (2)中获得的溶液合并混合均匀，过滤除菌，定量灌装于西林 瓶中， 真空冷冻干燥， 充入惰性气体， 压塞后扎盖， 得冻干粉针制剂产品。
13、 权利要求 12所述的方法， 其中步骤 (1)通过超滤进行过滤。
14、 权利要求 12或 13所述的方法， 其中步骤 (2)中活性炭的用量为 0.1~6.0g/100mL
15、 权利要求 12-14中任意一项所述的方法， 其中， 在步骤 (3)中， 经 0.05μιη~0.20μιη亚微米级微孔滤膜或荷电微孔滤膜过滤。
16、 权利要求 12所述的真空冷冻干燥方法， 其中， 在步骤 3 中， 制品预冻温度 -20°C至 -40°C， 时间 2-3 小时； 主干燥温度 -10 °C 6-10小时， 10°C 2-5小时， 20°C 2-5小时； 后干燥温度 30-50 °C， 4-10小时。
PCT/CN2009/074006 2008-09-17 2009-09-17 河豚毒素冻干粉针制剂及其制备方法 Ceased WO2010031346A1 (zh)

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CA2737463A CA2737463C (en) 2008-09-17 2009-09-17 Freeze-dried powder preparation of tetrodotoxin and the producing method thereof
US13/063,931 US20110201627A1 (en) 2008-09-17 2009-09-17 Freeze-dried reparation of tetrodotoxin and the producing method thereof
MX2011002924A MX2011002924A (es) 2008-09-17 2009-09-17 Preparacion de tetradotoxina de congelacion-deshidratacion y el metoo de producion de la misma.
EP09814066.8A EP2397143A4 (en) 2008-09-17 2009-09-17 FREEZE DRIED TETRODOTOXINE PREPARATION AND METHOD OF MANUFACTURING THEREOF
MX2011002851A MX2011002851A (es) 2008-09-17 2009-09-17 Preparacion de tetradotoxina de congelacion-deshidratacion y el metodo de produccion de la misma.
JP2011527192A JP2012502932A (ja) 2008-09-17 2009-09-17 フグ毒の凍結乾燥注射用粉末製剤及びその製造法
AU2009295083A AU2009295083B2 (en) 2008-09-17 2009-09-17 Freeze-dried preparation of tetrodotoxin and the producing method thereof
RU2011109869/15A RU2519654C2 (ru) 2008-09-17 2009-09-17 Лиофилизированный препарат на основе тетродотоксина и способ его производства
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CN107349206A (zh) * 2017-07-11 2017-11-17 东新皓特（北京）生化科技有限公司 河豚毒素在制备治疗新型毒品不良症状及复吸的药物组合物中的应用
CN110354003B (zh) * 2019-07-08 2022-05-27 广州贝研生物科技有限公司 传明酸作为冻干粉赋形剂的应用及冻干工艺
CN113419015A (zh) * 2021-07-26 2021-09-21 国家食品安全风险评估中心 一种基于河鲀鱼基料的天然河豚毒素标准样品制备方法及用途

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