CN101891751B - Method for preparing tetrodotoxin - Google Patents
Method for preparing tetrodotoxin Download PDFInfo
- Publication number
- CN101891751B CN101891751B CN2010102279942A CN201010227994A CN101891751B CN 101891751 B CN101891751 B CN 101891751B CN 2010102279942 A CN2010102279942 A CN 2010102279942A CN 201010227994 A CN201010227994 A CN 201010227994A CN 101891751 B CN101891751 B CN 101891751B
- Authority
- CN
- China
- Prior art keywords
- tetraodotoxin
- preparation
- tetrodotoxin
- temperature
- wash
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Medicinal Preparation (AREA)
Abstract
The invention relates to a tetrodotoxin preparation for treating drug addiction and relieving pain. The preparation comprises small-dose tetrodotoxin serving as a medicinal active ingredient and a pharmaceutical permissible cosolvent or support agent, wherein the cosolvent is amino acid. The amino acid is used as the cosolvent, so the amino acid has the function of the cosolvent, increases the water solubility of the tetrodotoxin, and can also form highly water-soluble salt solution with the tetrodotoxin. The tetrodotoxin used in the invention is prepared by a specific method; and the purity of the prepared tetrodotoxin reaches 98 percent and the yield is high. The preparation consisting of the tetrodotoxin prepared by the method and the amino acid has the advantages of high water solubility, high stability, safety, reliability, stable quality, no need of preservation in a refrigerator at a specific temperature, storage life of more than two years at room temperature, capability of greatly saving the storage and transportation cost and facilitating medical use, obvious treatment effect, and cure rate of 100 percent when particularly used for physiological detoxification.
Description
The application is dividing an application of Chinese patent application 200810127045.X.
Original application day: 2008-6-18
Original applying number: 200810127045.X
Original application invention and created name: a kind of drug rehabilitation, analgesic tetrodotoxin formulation of being used for
Technical field
The invention belongs to field of medicine preparations, relate in particular to a kind of drug rehabilitation, analgesic tetrodotoxin formulation of being used for.
Background technology
(Tetrodotoxin TTX) is the lower molecular weight alkaloids substance that a kind of effluent filefish internal organ extract to tetraodotoxin.Because its unique chemical structure makes it become the single-minded blocker of cellular sodium ionic channel.Therefore it has irreplaceable position in the biological study field as biological reagent.Tetraodotoxin shown pharmacotoxicological effect on clinical medicine just has been useful on the report of the disconnected alkaloids habituation venereal disease disease of clinical Jie as far back as the thirties in last century.Aspect analgesia therapy, also constantly there is report to release.Therefore, no matter it has all shown crucial status in the biological study field or in the clinical medicine application facet.
TTX be owing to just can block cellular sodium ionic passage under the situation of denier, and to not influence of potassium-channel, has the transitivity of height, therefore is that very useful nervous physiology research is used the instrument medicine.
Proved on the clinical medicine that TTX is good sedative agent, spasmolytic, analgesic agent, especially pernicious pain such as cancer had good analgesic activity, though to compare the time of response long slightly with anodynes such as traditional dolantin, morphines, its analgesic activity can reach 12-20 hour, and does not have habituation.Recently TTX also demonstrates good effect aspect drug rehabilitation, and general drug abstainer only needs to inject every day 10 μ gTTX, one pin, and 5-7 days is a course of treatment, and drug addiction can be controlled, and can alleviate the drug rehabilitation syndromes that usually occurs in the general drug rehabilitation greatly.
But tetraodotoxin injection liquid (aqueous solution) is very sensitive to temperature, is subjected to Temperature Influence easily and degrades, and temperature is high more, and it is fast more to degrade.In case being lower than the relative content of 90% or related substance of its labelled amount, effective active matter tetraodotoxin content just do not meet clinical service requirements above medical standard code limit (greater than contrast solution main peak area).Evidence, tetraodotoxin injection liquid (aqueous injection) are at room temperature unstable, in order to guarantee the quality of tetraodotoxin injection liquid, prevent that tetraodotoxin content from descending and its related substances raises, and must be placed in the 4-8 ℃ of refrigerator and preserve.This brings many difficulties and inconvenience just for clinical being suitable for of tetraodotoxin, correspondingly all must note keeping 4-8 ℃ of low temperature in storage, transportation, loading and unloading, wholesale, retail, hospital and each link of application, otherwise temperature drift may influence clinical effectiveness, to this, must manage to be solved as early as possible, develop safety and stability, Reliable Products that just can long storage under the room temperature.In order to address the above problem, a simple way is that tetraodotoxin is made freeze-dried preparation.Generally unsettled biologically active substance all can prolong in freeze-drying dehydration back storage period in the aqueous solution, when treating clinical application, adds sterilized water for injection regeneration and gets final product.But when tetraodotoxin is used for medicine, the consumption of its every dosage has only 0.5-60 μ g, so the tetraodotoxin of trace does not form desired solid residue after the freeze-drying dehydration in solution, the pharmaceutically receptible vehicle of essential adding, just the support that can provide micro-tetraodotoxin to be adhered to can form solids after solution freeze-drying dehydration.
Application number is that 03146020.8 Chinese patent application discloses a kind of composition and preparation method that containing of good stability meets under the room temperature that the human body safety injection uses freeze dried preparation of tetraodotoxin that can long storage that have, be by in tetrodotoxin formulation, adding a certain amount of disaccharide such as lactose, sucrose, cellobiose, maltose, making the freeze-dried problem that solves tetraodotoxin stability in preparation after the lyophilize.This is freeze-dried at room temperature store 1 year after, considerable change does not all take place in the content of its tetraodotoxin and its related substances, meets pharmaceutical required standard.
But because the biologically active substance tetraodotoxin is water-soluble hardly, although added a certain amount of solubility promoter in the composition of the disclosed tetraodotoxin freeze-dried preparation of above-mentioned patent application, the solubility of its freeze-dried preparation is still not so good.
In order to obtain the tetraodotoxin aqueous solution of desired concn, in the prior art, with the tetraodotoxin is that raw material is prepared into the tetraodotoxin salt derivative of highly water-soluble with it, is that 200410102449.5 Chinese patent application discloses various derivative of tetradotoxin that are prepared into salt form of tetraodotoxin (comprising tartrate, maleate, mesylate, hydrochloride, vitriol, hydrobromate, the acetate of tetraodotoxin, the equivalent derivative of citrate) and preparation method thereof as application number.
Chinese patent 02117928.X discloses a kind of drug rehabilitation, analgesic tetrodotoxin respiratory tract administration preparation of being used for, be characterized in that with tetraodotoxin citrate or tetraodotoxin acetate be bulk drug, make aerosol, sprays or aerosol preparations according to a conventional method.Respiratory tract administration preparation safety of the present invention, efficient, non-habituation, easy to use.Tetrodotoxin respiratory tract administration preparation of the present invention reaches more than 97% cancer pain patient is efficient, quits drug abuse efficiently to reach 100%, and be a kind of good drug rehabilitation, analgesic pharmaceutical preparation therefore.But its administering mode is limited to respiratory tract administration, comprises nasal cavity, oral cavity or tongue administration, preferably intranasal administration.Yet the specific absorption of nasal membrane is subjected to influence of various factors.And used acid is buffer reagent among the embodiment of this patent, and preparation illustrates that with the mole number of acid most TTX still do not form salt, and promptly most TTX still can not form the solution of highly water-soluble, fail fundamentally to solve its water miscible problem.
As seen, solve tetraodotoxin water-soluble and stable be the key issue for preparing tetrodotoxin formulation.
Summary of the invention
The purpose of this invention is to provide a kind of drug rehabilitation, analgesic tetrodotoxin formulation of being used for, said preparation is not only water-soluble good, and have good stability, safe and reliable, steady quality does not need to preserve in the refrigerator under the specified temp, and room temperature can reach more than 2 years following storage period, saved storage, transportation cost greatly, convenient medical treatment is used.
For achieving the above object, the present invention adopts following technical scheme:
A kind of drug rehabilitation, analgesic tetrodotoxin formulation of being used for is characterized in that, described preparation comprises as the low dose of tetraodotoxin of medicament active composition and be selected from solubility promoter or the caffolding agent that pharmaceutically allows that described solubility promoter is an amino acid.
The biologically active substance tetraodotoxin is water-soluble hardly, in order to obtain the aqueous solution of desired concn, in the prior art, with the tetraodotoxin is that raw material is prepared into the tetraodotoxin salt derivative of highly water-soluble with it, as the equivalent derivative of tartrate, maleate, mesylate, hydrochloride, vitriol, hydrobromate, acetate, citrate.But the inventor finds in actual applications, when preparing the tetraodotoxin salt derivative of these highly water-solubles, used acid is buffer reagent, most TTX still do not form salt, fail fundamentally to solve the solubility problem of tetraodotoxin, and these sour majorities are strong acid, can the toxicity of tetraodotoxin be damaged.The inventor finds pleasantly surprisedly that after having carried out a large amount of tests amino acid not only can play the effect of solubility promoter, can also form the TTX amino acid salts with tetraodotoxin, thereby form the salts solution of highly water-soluble, water-soluble good with the preparation of TTX amino acid salts preparation, preparation stability is also fine.
Among the present invention, described low dose of tetraodotoxin is that 5-20 μ g tetraodotoxin is contained in every preparation unit.
Among the present invention, described preparation unit is meant the pharmaceutically dosage forms unit of conventional formulation such as every of tablet, every capsules of capsule, granule every bag.
Among the present invention, preferred preparation way has following two kinds:
(1) described preparation is for being contained the injection of 5-20 μ g tetraodotoxin by every preparation unit of tetraodotoxin, solubility promoter and water for injection preparation.
(2) described preparation is for being contained the lyophilized injectable powder of 5-20 μ g tetraodotoxin by every preparation unit of tetraodotoxin, solubility promoter, caffolding agent and water for injection preparation.
The method preparation that tetraodotoxin injection provided by the present invention adopts those skilled in the art to use always, the following method preparation of preferred employing: in TTX, add solubility promoter, add water for injection, heating for dissolving again, be diluted to 1000ml, filter filtrate ultrafiltration, can, seal and make 1000, sterilization, check, promptly.
The method preparation that tetraodotoxin lyophilized injectable powder provided by the present invention adopts those skilled in the art to use always is preferably adopted following method preparation: add solubility promoter in TTX, add caffolding agent and water for injection again, heating and filtering, the filtrate ultrafiltration, packing, the lyophilize rear pressing cover is promptly.
Above-mentioned lyophilize is divided into four-stage: (1) pre-freeze 3-4 hour, temperature was at-30～-45 ℃; (2) drying under reduced pressure is 14 hours, and temperature is at-30～-45 ℃; (3) heated up dry 4 hours, temperature-15～-10C; (4) secondary temperature elevation is dry 4 hours, and temperature is at 30 ℃.
The present invention adopts distinctive low-temperature quick-freezing, the exsiccant freeze drying process that heats up stage by stage, the freeze-drying of gained tetraodotoxin is shaped good, has obtained the good crystal formation of the more conventional freeze-drying crystallization of stability, favourable stable transportation and storage have thoroughly solved the stable problem of tetraodotoxin lyophilized injectable powder.In a word, freeze drying process of the present invention has improved the stability of tetraodotoxin lyophilized injectable powder in preparation process greatly, and the tetraodotoxin lyophilized injectable powder quality stable homogeneous of preparation gained is stored and transport convenient.
Among the present invention, described solubility promoter is amino acid or its hydrochloride, preferred aspartic acid, arginine, glycine, L-glutamic acid, Methionin, ornithine, network propylhomoserin, Xie Ansuan, Histidine or L-cysteine hydrochloride, more preferably aspartic acid.Select for use amino acid or its hydrochloride as solubility promoter among the present invention, not only increased the solvability of tetraodotoxin in water, can also form the TTX amino acid salts with tetraodotoxin, thereby form the salts solution of highly water-soluble, but also replenish the necessary amino acid of human body simultaneously.
Among the present invention, described caffolding agent is N.F,USP MANNITOL, N.F,USP MANNITOL-glucose, N.F,USP MANNITOL-dextran, glucose or dextran, preferred N.F,USP MANNITOL-glucose, N.F,USP MANNITOL-dextran are more preferably than being N.F,USP MANNITOL-glucose of 5: 1 or N.F,USP MANNITOL-dextran of 4: 1.The inventor finds to select for use N.F,USP MANNITOL-glucose, N.F,USP MANNITOL-dextran can solve the stability problem of tetraodotoxin in preparation better when preparation is freeze-dried.
Among the present invention, described tetraodotoxin adopts following method preparation:
1) ovary of filefish or liver are rubbed, homogenate, stir with the alcoholic solution that contains acetate and to soak, soak solution is filtered, filtrate decompression reclaims alcohol, to there not being the alcohol flavor, crude venom;
2) will go up centrifugal, the filtration of crude venom that the step obtains, get filtrate; Filter residue merges to washings in the filtrate with containing the washing of 3～5% acetic acid water solution, leaves standstill, and separates water layer and upper strata oil reservoir, and oil reservoir is placed and is used for refining Vermiculated puffer liver oil;
3) will go up the water layer in step and filter, filtrate is crossed the weakly acidic cation-exchange resin post, and wash-out is collected elutriant;
4) with the elutriant concentrating under reduced pressure, the concentrated solution of gained is crossed the glucose gel post, and wash-out is collected elutriant, merges concentrating under reduced pressure, gets concentrated solution;
5) use the ammoniacal liquor adjust pH to 8-9 concentrated solution, after placing 45-50 hour under 5～10C temperature, sedimentation and filtration, washing, drying, refining, the cut at enrichment TTX peak, concentrating under reduced pressure, lyophilize are promptly.
In the prior art, tetraodotoxin also belongs to the crude product of toxin stoste type in a lot of tetrodotoxin formulations, and the purity of its tetraodotoxin is not high enough, directly influences the quality and the curative effect of its preparation.Adopt specific method to extract tetraodotoxin among the present invention, the tetraodotoxin purity height of gained not only, and be the bulk drug prepared preparation with it, good stability, solvability is good, and is evident in efficacy.
Extract after adopting the alcoholic solution that contains acetate to soak among the present invention, the extraction yield of tetraodotoxin is higher again.
Above-mentioned steps 1) immersion described in is to contain the 30%-100% methyl alcohol of 3～5% acetic acid or ethanolic soln to stir and soak 3～6 times.Select the 30%-100% methyl alcohol or the alcohol solution dipping that contain 3～5% acetic acid among the present invention for use, more help the dissolving of TTX, reduced macro-molecular protein simultaneously and dissolved in, therefore, can avoid the heat isolating protein.
Above-mentioned steps 2) washing described in is washing 2-4 time, preferred 3 times; Leaving standstill under 5-15 ℃ of temperature step 2) placed 8-12 hour.
Above-mentioned steps 3) wash-out in is first water wash-out, uses 10% acetic acid wash-out again; Wash-out in the step 4) is for using the 50% ethanolic soln wash-out that contains 5% acetic acid.Among the present invention, used elutriant difference in two steps, one side are to select different eluents according to the filler of the post of twice usefulness is different, then are to adopt type of elution elute effect provided by the present invention good on the other hand.
The used glucose gel post of the present invention is this area glucose gel post commonly used, preferred LH-20 glucose gel post.
With respect to prior art, the present invention has following advantage:
(1) drug rehabilitation, the analgesic tetrodotoxin formulation of being used for provided by the present invention, not only water-soluble good, and have good stability, safe and reliable, steady quality does not need to preserve in the refrigerator under the specified temp, and room temperature can reach more than 2 years following storage period, saved storage, transportation cost greatly, convenient medical treatment is used;
(2) tetraodotoxin bulk drug provided by the present invention, purity are greater than 98%, and it is water-soluble good, the prepared preparation excellent in stability, and good effect, result of treatment is obvious;
(3) preparation method of tetraodotoxin provided by the present invention prepare tetraodotoxin to the destruction of tetraodotoxin less, loss is few, yield is high, purity is high, good stability;
(4) need not adopt charcoal absorption among the preparation method of tetraodotoxin provided by the present invention, adopt column chromatography twice, can not only remove impurity effectively, also reduce extraction time, the biological activity loss of tetraodotoxin is minimized;
(5) keep lesser temps to handle the destruction of having avoided temperature that tetraodotoxin is caused in the extraction separation process among the preparation method of tetraodotoxin provided by the present invention;
(6) oil reservoir behind the extraction separation can also be used for refining Vermiculated puffer liver oil among the preparation method of tetraodotoxin provided by the present invention, removes poison and greasy residue, can be used as the feed of poultry after fermenting, thereby has avoided unnecessary waste and environmental pollution;
Description of drawings
Fig. 1 is for determining tetraodotoxin lowest total of the melting point curve synoptic diagram;
Fig. 2 is the freeze-drying curve synoptic diagram.
Embodiment
Below be the specific embodiment of the present invention, described embodiment is in order to further describe the present invention, rather than restriction the present invention.
The preparation of [embodiment 1] bulk drug tetraodotoxin
Ovary 100kg with filefish, mincer rubs, and with refiner homogenate, adds 100 liters of 60% ethanol that contain 3% acetic acid, stir and soak 3 times, filtrate, filtration under diminished pressure are again got in filtration, centrifuging 4 times, filtrate is by D152 type acidulous cation resin post, to remove dried up wash-out pillar, be colourless to elutriant, use 10% aqueous acetic acid wash-out (elution speed 50ml/ branch) then.Detect with HPLC, enrichment contains venom, merges concentrating under reduced pressure, and concentrated solution is by glucose gel LH-20 post, to contain 50% ethanol liquid wash-out of 5% acetic acid, HPLC detects, and enrichment contains the elutriant of TTX, merging, concentrating under reduced pressure, enriched material is transferred pH=8.0 with ammoniacal liquor, low temperature was placed 48 hours for 5 °, with sedimentation and filtration, washing, drying, got white depositions TTX.
HPLC is refining with preparation, the cut at enrichment TTX peak, and concentrating under reduced pressure gets concentrated solution, after the lyophilize, gets the toxin 2.0g of TTX, purity 〉=98%.
The preparation of [embodiment 2] bulk drug tetraodotoxin
Ovary 100kg with filefish, mincer rubs, and with refiner homogenate, adds 120 liters of 50% methyl alcohol that contain 5% acetic acid, stir and soak 6 times, filtration, centrifuging are got filtrate 4 times, filtration under diminished pressure, filtrate is by D152 type acidulous cation resin post, to remove dried up wash-out pillar, be colourless to elutriant, use 10% acetate solution wash-out (elution speed 50ml/ branch) then.Detect with HPLC, enrichment contains venom, merging, concentrating under reduced pressure, concentrated solution is by glucose gel LH-20 post, to contain 50% ethanol liquid wash-out of 5% acetic acid, HPLC detects, the elutriant that enrichment contains TTX merges, concentrating under reduced pressure, and enriched material is transferred pH9.0 with ammoniacal liquor, and low temperature was placed 48 hours for 10 ℃, filter, do bath, get white depositions TTX.
HPLC is refining with preparation, the cut at enrichment TTX peak, and concentrating under reduced pressure gets concentrated solution, and lyophilize gets TTX toxin 2.0g, purity 〉=98%.
The preparation of [embodiment 3] bulk drug tetraodotoxin
Get the liver 100kg of filefish, mincer rubs, and with refiner homogenate, adds 110 liters of 60% ethanol that contain 4% acetic acid, stir and soak 4 times, filtrate, filtration under diminished pressure are again got in filtration, centrifuging 4 times, filtrate is by D152 type acidulous cation resin post, to remove dried up wash-out pillar, be colourless to elutriant, use 10% aqueous acetic acid wash-out (elution speed 50ml/ branch) then.Detect with HPLC, enrichment contains venom, merges concentrating under reduced pressure, and concentrated solution is by glucose gel LH-20 post, to contain 50% ethanol liquid wash-out of 5% acetic acid, HPLC detects, and enrichment contains the elutriant of TTX, merging, concentrating under reduced pressure, enriched material is transferred pH=8.5 with ammoniacal liquor, low temperature was placed 48 hours for 8 ℃, with sedimentation and filtration, washing, drying, got white depositions TTX.
HPLC is refining with preparation, the cut at enrichment TTX peak, and concentrating under reduced pressure gets concentrated solution, after the lyophilize, gets the toxin 2.0g of TTX, purity 〉=98%.
The preparation of [embodiment 4] bulk drug tetraodotoxin
1) get the liver 100g of filefish, mincer rubs, and with refiner homogenate, adds 115 liters of 30% methanol solutions that contain 3% acetic acid and stirs and soak 2 times, and soak solution is filtered, and filtrate decompression reclaims alcohol, to there not being the alcohol flavor, gets crude venom;
2) will go up centrifugal, the filtration of crude venom that the step obtains, get filtrate; Filter residue merges to washings in the filtrate with containing 3% acetic acid water solution washing 2 times, leaves standstill 8 hours under 5 ℃ of temperature, separates water layer and upper strata oil reservoir, and oil reservoir is placed and is used for refining Vermiculated puffer liver oil;
3) will go up the water layer in step and filter, filtrate is crossed D152 type weakly acidic cation-exchange resin post, and first water wash-out is used 10% acetic acid wash-out again, collects elutriant;
4) with the elutriant concentrating under reduced pressure, the concentrated solution of gained is crossed LH-20 glucose gel post, with the 50% ethanolic soln wash-out that contains 5% acetic acid, collects elutriant, merges concentrating under reduced pressure, gets concentrated solution;
5) with concentrated solution with ammoniacal liquor adjust pH to 8, after placing 45 hours under 5 ℃ of temperature, sedimentation and filtration, washing, drying, refining, the cut at enrichment TTX peak, concentrating under reduced pressure, lyophilize promptly, purity 〉=98%.
The preparation of [embodiment 5] bulk drug tetraodotoxin
1) get the ovary 100g of filefish, mincer rubs, and with refiner homogenate, adds 110 liters of 100% ethanolic solns that contain 5% acetic acid and stirs and soak 4 times, and soak solution is filtered, and filtrate decompression reclaims alcohol, to there not being the alcohol flavor, gets crude venom;
2) will go up centrifugal, the filtration of crude venom that the step obtains, get filtrate; Filter residue merges to washings in the filtrate with containing 5% acetic acid water solution washing 4 times, leaves standstill 12 hours under 15 ℃ of temperature, separates water layer and upper strata oil reservoir, and oil reservoir is placed and is used for refining Vermiculated puffer liver oil;
3) will go up the water layer in step and filter, filtrate is crossed D152 type weakly acidic cation-exchange resin post, and first water wash-out is used 10% acetic acid wash-out again, collects elutriant;
4) with the elutriant concentrating under reduced pressure, the concentrated solution of gained is crossed LH-20 glucose gel post, with the 50% ethanolic soln wash-out that contains 5% acetic acid, collects elutriant, merges concentrating under reduced pressure, gets concentrated solution;
5) with concentrated solution with ammoniacal liquor adjust pH to 9, after placing 50 hours under 15 ℃ of temperature, sedimentation and filtration, washing, drying, refining, the cut at enrichment TTX peak, concentrating under reduced pressure, lyophilize promptly, purity 〉=98%.
The preparation of [embodiment 6] bulk drug tetraodotoxin
1) get the liver 100g of filefish, mincer rubs, and with refiner homogenate, adds 115 liters of 60% ethanolic solns that contain 3% acetic acid and stirs and soak 2 times, and soak solution is filtered, and filtrate decompression reclaims alcohol, to there not being the alcohol flavor, gets crude venom;
2) will go up centrifugal, the filtration of crude venom that the step obtains, get filtrate; Filter residue merges to washings in the filtrate with containing 3% acetic acid water solution washing 3 times, leaves standstill 8 hours under 5 ℃ of temperature, separates water layer and upper strata oil reservoir, and oil reservoir is placed and is used for refining Vermiculated puffer liver oil;
3) will go up the water layer in step and filter, filtrate is crossed D152 type weakly acidic cation-exchange resin post, and first water wash-out is used 10% acetic acid wash-out again, collects elutriant;
4) with the elutriant concentrating under reduced pressure, the concentrated solution of gained is crossed LH-20 glucose gel post, with the 50% ethanolic soln wash-out that contains 5% acetic acid, collects elutriant, merges concentrating under reduced pressure, gets concentrated solution;
5) with concentrated solution with ammoniacal liquor adjust pH to 8, after placing 45 hours under 10 ℃ of temperature, sedimentation and filtration, washing, drying, refining, the cut at enrichment TTX peak, concentrating under reduced pressure, lyophilize promptly, purity 〉=98%.
The preparation of [example of formulations 1] tetraodotoxin lyophilized injectable powder
5.0mgTTX add the 5.0g arginine, add the dissolving of 5.0g N.F,USP MANNITOL, add water for injection, heating for dissolving is diluted to 1000ml, filtration, filtrate ultrafiltration, packing, lyophilize, rear pressing cover finishes.Make 1000, every contains TTX5.0 μ g.
Wherein, above-mentioned lyophilize is divided into four-stage: (1) pre-freeze 3 hours, and temperature is at-30 ℃; (2) drying under reduced pressure is 14 hours, and temperature is at-30 ℃; (3) heated up dry 4 hours, temperature is at-15 ℃; (4) secondary temperature elevation is dry 4 hours, and temperature is at 30 ℃.
The preparation of [example of formulations 2] tetraodotoxin lyophilized injectable powder
5.0mgTTX add the 5.0g glycine, add 5.0g N.F,USP MANNITOL-glucose (5: 1), add water for injection, heating for dissolving is diluted to 1000ml, filtration, filtrate ultrafiltration, packing, lyophilize, rear pressing cover finishes.Make 1000, every contains TTX5.0 μ g.
Above-mentioned lyophilize is divided into four-stage: (1) pre-freeze 4 hours, and temperature is at-45 ℃; (2) drying under reduced pressure is 14 hours, and temperature is at-45 ℃; (3) heated up dry 4 hours, temperature is at-10 ℃; (4) secondary temperature elevation is dry 4 hours, and temperature is at 30 ℃.
The preparation of [example of formulations 3] tetraodotoxin lyophilized injectable powder
10.0mg TTX adds 5.0g L-glutamic acid, adds 5.0g N.F,USP MANNITOL-glucose (5: 1), adds water for injection, heating for dissolving is diluted to 1000ml, filtration, filtrate ultrafiltration, and packing, lyophilize, rear pressing cover finishes.Make 1000, every contains TTX10.0 μ g.
Above-mentioned lyophilize is divided into four-stage: (1) pre-freeze 3.5 hours, and temperature is at-35 ℃; (2) drying under reduced pressure is 14 hours, and temperature is at-35 ℃; (3) heated up dry 4 hours, temperature is at-12 ℃; (4) secondary temperature elevation is dry 4 hours, and temperature is at 30 ℃.
The preparation of [example of formulations 4] tetraodotoxin lyophilized injectable powder
10.0mgTTX add 5.0g Methionin, add 5.0g glucose, add water for injection, heating for dissolving is diluted to 1000ml, filtration, filtrate ultrafiltration, packing, lyophilize, rear pressing cover finishes.Make 1000, every contains TTX10.0 μ g.
Above-mentioned lyophilize is divided into four-stage: (1) pre-freeze 3.8 hours, and temperature is at-40 ℃; (2) drying under reduced pressure is 14 hours, and temperature is at-40 ℃; (3) heated up dry 4 hours, temperature is at-13 ℃; (4) secondary temperature elevation is dry 4 hours, and temperature is at 30 ℃.
The preparation of [example of formulations 5] tetraodotoxin lyophilized injectable powder
10.0mgTTX add 5.0gL-cysteine hydrochloride, add 5.0g glucose, add water for injection, heating for dissolving is diluted to 1000ml, filtration, filtrate ultrafiltration, packing, lyophilize, rear pressing cover finishes.Make 1000, every contains TTX10.0 μ g.
Above-mentioned lyophilize is divided into four-stage: (1) pre-freeze 3-4 hour, temperature was at-30～-45 ℃; (2) drying under reduced pressure is 14 hours, and temperature is at-30～-45 ℃; (3) heated up dry 4 hours, temperature is at-15～-10 ℃; (4) secondary temperature elevation is dry 4 hours, and temperature is at 30 ℃.
The preparation of [example of formulations 6] tetraodotoxin lyophilized injectable powder
15.0mgTTX add 5.0g L-glutamic acid, add the 5.0g dextran, add water for injection, heating for dissolving is diluted to 1000ml, filtration, filtrate ultrafiltration, packing, lyophilize, rear pressing cover finishes.Make 1000, every contains TTX15.0 μ g.
Above-mentioned lyophilize is divided into four-stage: (1) pre-freeze 3.6 hours, and temperature is at-33 ℃; (2) drying under reduced pressure is 14 hours, and temperature is at-36 ℃; (3) heated up dry 4 hours, temperature is at-13 ℃; (4) secondary temperature elevation is dry 4 hours, and temperature is at 30 ℃.
The preparation of [example of formulations 7] tetraodotoxin lyophilized injectable powder
15.0mgTTX add 5.0g L-glutamic acid, add 5.0g N.F,USP MANNITOL, add water for injection, heating for dissolving is diluted to 1000ml, filtration, filtrate ultrafiltration, packing, lyophilize, rear pressing cover finishes.Make 1000, every contains TTX15.0 μ g.
Above-mentioned lyophilize is divided into four-stage: (1) pre-freeze 3.6 hours, and temperature is at-33 ℃; (2) drying under reduced pressure is 14 hours, and temperature is at-36 ℃; (3) heated up dry 4 hours, temperature is at-13 ℃; (4) secondary temperature elevation is dry 4 hours, and temperature is at 30 ℃.
The preparation of [example of formulations 8] tetraodotoxin lyophilized injectable powder
20.0mgTTX add the 5.0g glycine, add 5.0g N.F,USP MANNITOL-glucose (5: 1), add water for injection, heating for dissolving is diluted to 1000ml, filtration, filtrate ultrafiltration, packing, lyophilize, rear pressing cover finishes.Make 1000, every contains TTX20.0 μ g.
Above-mentioned lyophilize is divided into four-stage: (1) pre-freeze 3-4 hour, temperature was at-30～45 ℃; (2) drying under reduced pressure is 14 hours, and temperature is at-30～-45 ℃; (3) heated up dry 4 hours, temperature is at-15～-10 ℃; (4) secondary temperature elevation is dry 4 hours, and temperature is at 30 ℃.
The preparation of [example of formulations 9] tetraodotoxin lyophilized injectable powder
20.0mgTTX add the 5.0g arginine, add 5.0g N.F,USP MANNITOL-glucose (5: 1), add water for injection, heating for dissolving is diluted to 1000ml, filtration, filtrate ultrafiltration, packing, lyophilize, the rear pressing cover that finishes is made 1000.Add the water for injection heating for dissolving, be diluted to 1000ml,, filter, the filtrate ultrafiltration, the filtrate can is sealed and is made 1000, and every contains TTX20.0 μ g.
Above-mentioned lyophilize is divided into four-stage: (1) pre-freeze 3.5 hours, and temperature is at-35 ℃; (2) drying under reduced pressure is 14 hours, and temperature is at-35 ℃; (3) heated up dry 4 hours, temperature is at-12 ℃; (4) secondary temperature elevation is dry 4 hours, and temperature is at 30 ℃.
The preparation of [example of formulations 10] tetraodotoxin lyophilized injectable powder
5.0mgTTX add the 5.0g aspartic acid, add 5.0g N.F,USP MANNITOL-glucose (5: 1), add water for injection, heating for dissolving is diluted to 1000ml, filtration, filtrate ultrafiltration, packing, lyophilize, rear pressing cover finishes.Make 1000, every contains TTX5.0 μ g.
Above-mentioned lyophilize is divided into four-stage: (1) pre-freeze 4 hours, and temperature is at-45 ℃; (2) drying under reduced pressure is 14 hours, and temperature is at-45 ℃; (3) heated up dry 4 hours, temperature is at-10 ℃; (4) secondary temperature elevation is dry 4 hours, and temperature is at 30 ℃.
The preparation of [example of formulations 11] tetraodotoxin injection
5.0mgTTX add 50mg L-glutamic acid, add water for injection, heating for dissolving is diluted to 1000ml, filters, the filtrate ultrafiltration, the filtrate can is sealed and is made 1000, sterilization, check.Every contains TTX5.0 μ g.The preparation of [example of formulations 12] tetraodotoxin injection
5.0mgTTX add the 50mg arginine, add water for injection, heating for dissolving is diluted to 1000ml,, filter, the filtrate ultrafiltration, the filtrate can is sealed and is made 1000, sterilization, check.Every contains TTX5.0 μ g.The preparation of [example of formulations 13] tetraodotoxin injection
10.0mgTTX add the 50mg glycine, add water for injection, heating for dissolving is diluted to 1000ml,, filter, the filtrate ultrafiltration, the filtrate can is sealed and is made 1000, sterilization, check.Every contains TTX10.0 μ g.
The preparation of [example of formulations 14] tetraodotoxin injection
10.0mgTTX add the 50mg arginine, add water for injection, heating for dissolving is diluted to 1000ml, filters, the filtrate ultrafiltration, the filtrate can is sealed and is made 1000, sterilization, check.Every contains TTX10.0 μ g.
The preparation of [example of formulations 15] tetraodotoxin injection
15mgTTX adds the 75mg glycine, adds water for injection, and heating for dissolving is diluted to 1000ml, filter, and the filtrate ultrafiltration, the filtrate can is sealed and is made 1000, sterilization, check.Every contains TTX15.0 μ g.
The preparation of [example of formulations 16] tetraodotoxin injection
15mgTTX adds 75mg L-glutamic acid, adds water for injection, and heating for dissolving is diluted to 1000ml, filter, and the filtrate ultrafiltration, the filtrate can is sealed and is made 1000, sterilization, check.Every contains TTX15.0 μ g.
The preparation of [example of formulations 17] tetraodotoxin injection
20mgTTX adds 100mg Methionin, adds water for injection, and heating for dissolving is diluted to 1000ml, filter, and the filtrate ultrafiltration, the filtrate can is sealed and is made 1000, sterilization, check.Every contains TTX20.0 μ g.
The preparation of [example of formulations 18] tetraodotoxin injection
10mgTTX adds 50mg L-cysteine hydrochloride, adds water for injection, and heating for dissolving is diluted to 1000ml, filter, and the filtrate ultrafiltration, the filtrate can is sealed and is made 1000, sterilization, check.Every contains TTX10.0 μ g.
Below come the beneficial effect of product of the present invention is described in detail by testing example.
[test example 1] stability test
Get the prepared tetraodotoxin lyophilized injectable powder of example of formulations 1, according to " two appendix XIX of Chinese pharmacopoeia version in 2005 C medicine stability test governing principle has been carried out influence factor test, accelerated test and test of long duration.The investigation project comprises: proterties, discriminating, content.
(1) influence factor test
1, exposure experiments to light
Instrument: ST-80C luxmeter, fluorescent lamp
Condition: 4500 ± 500LX
With the sample of example of formulations 1, lot number 20040906 is put under the fluorescent lamp, respectively at 5 days, the 10 days every relevant indexs of sampling and measuring, the results are shown in Table 1.
Table 1. exposure experiments to light
2, high temperature test
Instrument: the medical loft drier of AXGZ-10 type
Get the sample of example of formulations 1, lot number 20040906 is put under 60 ℃ of conditions, respectively at 5 days, the 10 days every relevant indexs of sampling and measuring, the results are shown in 2.
Table 2. high temperature test
Through test, this product is under the strong illumination condition, and test back content reduces.High temperature is in the time of 10 days, and content also has reduction obviously.Add wet test, because of the easy moisture absorption of the raw material of this product, so do not reexamine this order.Find out that by test-results the holding conditions of this product answers low temperature, shading, airtight preservation.
(2) low temperature is placed test for a long time
Sample: get the sample of example of formulations 1,2,3, lot number 20040906,20040919,20040928
Sample packaging: aluminum-plastic packaged (listing packing).
Test apparatus: climatic chamber
Test conditions: 2～6 ℃ of temperature
Get packaged sample, put into climatic chamber, respectively at 3,6,9,12,18,24,36 months every relevant indexs of sampling and measuring, measurement result saw Table 3.
Table 3. low temperature is placed test for a long time
The result shows, this product under the commercially available back condition, every index and relatively do not have considerable change in 0 day before and after the low-temperature test.
(3) normal temperature is placed test for a long time
Sample: get the sample of example of formulations 1,2,3, lot number 20040906,20040919,20040928
Sample packaging: aluminum-plastic packaged (listing packing).
Test apparatus: climatic chamber
Condition: 30 ± 2 ℃ of temperature, relative humidity 60 ± 10%
Get listing packing sample, place the chamber that keeps sample, respectively at 3,6,9,12,18,24,36 months sampling and measuring, measurement result saw Table 4.
Table 4. normal temperature is placed test for a long time
The result shows, this product normal temperature test of long duration 36 months, and every index of three batch samples and 0 day every index relatively have no significant change.As seen, product tetrodotoxin formulation of the present invention has good stability.
The analgesic activity of [test example 2] product tetrodotoxin formulation of the present invention
1, mouse hot plate (55 ℃) analgesic test
Kunming mouse is provided by Military Medical Science Institute's animal center, meets national SPF level laboratory animal standard.♀, body weight 18～22g.Raise in cages 6～12 in every cage.20～24 ℃ of room temperatures, natural lighting, drinking water is not limit.With the mouse random packet, 15 every group.The positive control medicine: Srm-Rhotaard (morphine hydrochloride) is available from Qinghai Pharmaceutic Plant, lot number: 981101; Be subjected to the reagent thing: the tetraodotoxin lyophilized injectable powder of example of formulations 5.Lot number: 070906, provide by Shanghai YiNian Biology Science Co., Ltd.On hot plate pain threshold detector (GJ-8402 type hot plate pain threshold detector), carry out the hot-plate analgesic test.55 ℃ of hot plate temperatures, the pain reaction index is for licking metapedes, and dead line, (cut-off time) was 60s, measured the latent period of pain reaction.Analgesia efficiency calculation formula: analgesia efficient (%)=(latent period of the latent period-control group of experimental group)/(latent period of dead line-control group) * 100%.Treatment sequence is as follows: what subcutaneous injection gave various dose is subjected to reagent thing or positive control medicine, measures adding metapedes pain reaction latent period of mouse behind the 30min.Each group all is control group with the solvent.Experimental result is represented with mean ± standard error, adopts the SPSS statistical software to carry out variance analysis (ANOVA), relatively selects the LSD method between group for use, and calculates ED50 with the Bliss method.
Table 5. product tetrodotoxin formulation of the present invention stimulates the analgesic activity of (55 ℃) to the mouse heat injury
Annotate: mean ± standard error .*P＜0.05, * * P＜0.01, compare with the solvent control group * * * P＜0.001
Table 5 result shows that in mouse hot plate (55 ℃) analgesic test, the tetraodotoxin lyophilized injectable powder of mouse subcutaneous injection example of formulations 5 can produce significant analgesia role.The tetraodotoxin lyophilized injectable powder 1-16 μ g/kg of example of formulations 5 is dose-dependently and prolongs mouse licks the metapedes pain reaction on hot plate (55 ℃) latent period.From the low dosage to the high dosage, the analgesia efficient of the tetraodotoxin lyophilized injectable powder of example of formulations 5 is respectively 4.8%, 13.3%, 49.2%, 74.1% and 81.1%.The tetraodotoxin lyophilized injectable powder analgesic activity ED50 of mouse hot plate (55 ℃) method example of formulations 5 and 95% credibility interval are 4.9 (3.3-7.8) μ g/kg.[4.9 (2.2-8.1) mg/kg] compares with the morphine analgesia effect, and the intensity of the tetraodotoxin lyophilized injectable powder analgesic activity of example of formulations 5 approximately is 1000 times of morphine.According to the tetraodotoxin freeze-dried powder agent dose of example of formulations 5 in mouse hot plate (55 ℃) the method analgesic test and the relation of analgesia efficient, calculating the clinical effective dose scope is 0.36-0.85 μ g/kg.Research data shows that the hormesis of hot plate (55 ℃) pain is stronger.Therefore, the clinical effective dose of above-mentioned deduction is applicable to the moderate and severe pain patient.
The preparation of other example of formulations has similar result among the present invention in this test.
2, mouse acetic acid twisting analgesic test
Kunming mouse is provided by Military Medical Science Institute's animal center, meets national SPF level laboratory animal standard.Body weight 18～22g.Raise in cages, 6～12 in every cage, 20～24 ℃ of room temperatures, natural lighting, drinking water is not limit.With the mouse random packet, 16 every group, ♂ ♀ half and half.The positive control medicine: Srm-Rhotaard (morphine hydrochloride) is available from Qinghai Pharmaceutic Plant, lot number: 981101; Be subjected to the reagent thing: the tetraodotoxin lyophilized injectable powder of example of formulations 5.Lot number: 070906, provide by Shanghai YiNian Biology Science Co., Ltd.Test is an algogen with 0.6% acetic acid.Subcutaneous injection gives the tetraodotoxin lyophilized injectable powder (being subjected to the reagent thing) or the Srm-Rhotaard (positive control medicine) of the example of formulations 5 of various dose, with the negative contrast of solvent.Behind the 30min, mouse writhing number of times in the acetic acid 0.2ml of every abdominal injection 0.6%, immediate record 15min.With mouse waist muscle contraction repeatedly, hogback appear, buttocks reverses and hind leg is stretched to the writhing response positive.Administration group and group of solvents mouse writhing number are compared, and formula is calculated analgesia efficient.Calculation formula: analgesia efficient (%)=(control group turn round body number of times-experimental group turn round the body number of times)/control group turn round body number of times * 100%.Experimental result is represented with mean ± standard error, adopts the SPSS statistical software to carry out variance analysis (ANOVA), relatively selects the LSD method between group for use, and calculates ED50 with the Bliss method.
Table 6. product tetrodotoxin formulation of the present invention is to the analgesic activity of mouse chemical noxious stimulus (0.6% acetic acid)
Annotate: mean ± standard error .*P＜0.05, * * P＜0.01, compare with the solvent control group * * * P＜0.001
Table 6 result shows, turns round in the analgesic test of body at mouse acetic acid (0.6%), can obviously suppress the pain reaction that abdominal injection 0.6% acetic acid induced mice is turned round body for the tetraodotoxin lyophilized injectable powder of mouse subcutaneous injection example of formulations 5, is dose-dependently.The analgesia efficient of the tetraodotoxin lyophilized injectable powder of low dosage 0.5 μ g/kg example of formulations 5 is 13.8%, and high dosage 8 μ g/kg can reach 89.9%.(ED50 of the tetraodotoxin lyophilized injectable powder analgesic activity of 0.6 writhing method example of formulations 5 and 95% credibility interval are 1.9 (1.7～2.2) μ g/kg to mouse acetic acid, and the ED50 of morphine and 95% credibility interval are 1.0 (0.9～1.2) μ g/kg.By contrast, to turn round the action intensity of body pain reaction approximately be 526 times of morphine to the anti-mouse acetic acid of the tetraodotoxin lyophilized injectable powder of example of formulations 5 (0.6%).According to the tetraodotoxin freeze-dried powder agent dose of mouse acetic acid (0.6%) writhing method example of formulations 5 and the relation of analgesia efficient, calculating the clinical effective dose scope is 0.19～0.24 μ g/kg.Compare with hot plate (55 ℃) pain stimulation, the pain stimulation that mouse peritoneal is injected 0.6% acetic acid a little less than.Therefore, the clinical effective dose of above-mentioned deduction is applicable to the mild or moderate pain-suffered patient.
The preparation of other example of formulations has similar result among the present invention in this test.
3, the time-effect relationship of product tetrodotoxin formulation analgesic activity of the present invention
Kunming mouse is provided by Military Medical Science Institute's animal center, meets national SPF level laboratory animal standard.Body weight 18～22g.Raise in cages 6～12 in every cage. 20～24 ℃ of room temperatures, natural lighting, drinking water is not limit.With the mouse random packet, 10 every group, ♂ ♀ half and half.The positive control medicine: Srm-Rhotaard (morphine hydrochloride) is available from Qinghai Pharmaceutic Plant, lot number: 981101; Be subjected to the reagent thing: the tetraodotoxin lyophilized injectable powder of example of formulations 5.Lot number: 070906, provide by Shanghai YiNian Biology Science Co., Ltd.Test is an algogen with 0.6% acetic acid.Subcutaneous injection gives the tetraodotoxin lyophilized injectable powder of the example of formulations 5 of 4 μ g/kg.Respectively at 5,10,15,30,60,120,180,240,300, behind the 360min, mouse writhing number of times in the acetic acid 0.2ml of every abdominal injection 0.6%, immediate record 15min.With mouse waist muscle contraction repeatedly, hogback appear, buttocks reverses and hind leg is stretched to the writhing response positive.Administration group and group of solvents mouse writhing number are compared, and calculate analgesia efficient.Calculation formula: analgesia efficient (%)=(control group turn round body number of times-experimental group turn round the body number of times)/control group turn round body number of times * 100%.Experimental result is represented with mean ± standard error, adopts the SPSS statistical software to carry out variance analysis (ANOVA), relatively selects the LSD method between group for use.
Experimental result according to table 6, selecting analgesia efficient is the tetraodotoxin freeze-dried powder agent dose 4 μ g/kg of 75.9% example of formulations 5, adopts mouse acetic acid (0.6%) writhing method then to measure the time-effect relationship of the tetraodotoxin lyophilized injectable powder analgesic activity of example of formulations 5.The result of table 3 shows, gives the tetraodotoxin lyophilized injectable powder 4 μ g/kg of mouse subcutaneous injection example of formulations 5, and analgesic activity is the 10min onset after administration, reaches maximum analgesia efficient 72% behind the 60min.Sustainable 6 hours of the analgesic activity of the tetraodotoxin lyophilized injectable powder of example of formulations 5.
Table 7. product tetrodotoxin formulation of the present invention is turned round the time-effect relationship of body analgesic activity to mouse acetic acid (0.6%)
Annotate: mean ± standard error .P value and solvent control group are relatively.
The preparation of other example of formulations has similar result among the present invention in this test.
The drug treatment function of [test example 3] product tetrodotoxin formulation of the present invention
1, the morphine-dependent mice naloxone is urged the withdrawal and treatment test
Kunming mouse is provided by Military Medical Science Institute's animal center, meets national II level laboratory animal standard.Body weight 18～22g.Raise in cages 6～12 in every cage. 20～24 ℃ of room temperatures, natural lighting, drinking water is not limit.Medicine and reagent: be subjected to the reagent thing: the tetraodotoxin lyophilized injectable powder of example of formulations 5, lot number: 070906, provide by Shanghai YiNian Biology Science Co., Ltd.10 μ g/ml are made into desired concn with solvent; Srm-Rhotaard (morphinehydrochloride) is available from Qinghai Pharmaceutic Plant, lot number: 981101; Naloxone hydrochloride: Sigma company produces, and lot number: 42k1184 prepares with physiological saline.Route of administration and volume: subcutaneous injection, administration volume: 0.1ml/10g.Test method: mouse is divided into 5 groups at random, and 10 every group, half and half, 5 group of tetraodotoxin lyophilized injectable powder group that is respectively solvent control group, 4 example of formulations 5 of ♀ ♂.Each organizes according to the form below subcutaneous injection every day morphine 3 times, and (20:00), successive administration 8d, d8 early 8:00 are the administration of morphine last for 8:00,14:00.Set up morphine and rely on model.
Set up the treatment sequence table that morphine relies on model
Urged and giving up with naloxone hydrochloride (2mg/kg, 0.1ml/10g, abdominal injection) in 2 hours after the administration of morphine last, 30min treats administration before urging.Solvent control group subcutaneous injection solvent 0.1ml/10g; The tetraodotoxin lyophilized injectable powder of example of formulations 5 is subcutaneous injection 1,2,4,8 μ g/kg respectively.The number of skips in the mouse 10min of back urged in record, and urge give up before and urge body weight when giving up back 30min and 60min, the body weight before giving up with urgency is a base value, calculates the body weight change rate after urging.Calculation formula: body weight * 100% before body weight change rate (%)=(body weight before the body weight-urgency after the urgency)/urgency.Experimental result is represented with mean ± standard error, adopts the SPSS statistical software to carry out variance analysis (ANOVA), relatively selects the LSD method between group for use.
Table 8. product tetrodotoxin formulation of the present invention is to the influence of morphine-dependent mice withdrawal symptom
Annotate: mean ± standard error .*P＜0.05, * * P＜0.01, compare with solvent (0 μ g/kg) control group * * * P＜0.001.
The experimental result of table 8 shows: after solvent control gave naloxone (2mg/kg) urgency, giving up number of skips was 66.8 ± 9.74, and body weight all descended 2.7% after 30min and 60min were given up in urgency, illustrated that mouse has formed tangible drug dependence to morphine.Before the injection naloxone, give the tetraodotoxin lyophilized injectable powder of medicine example of formulations 5, be dose-dependent inhibition and give up jump behavior and weight loss, the tetraodotoxin lyophilized injectable powder 2 μ g/kg of example of formulations 5 can obviously alleviate and urge the weight loss give up behind the 30min, 4 μ g/kg and 8 μ g/kg obviously to reduce giving up number of skips and urge the weight loss of giving up behind the 60min.The tetraodotoxin lyophilized injectable powder that The above results shows example of formulations 5 has tangible detoxification treatment effect to the urgency Withrawal symptom of morphine-dependent mice.Urge the tetraodotoxin freeze-dried powder agent dose of withdrawal and treatment test preparation embodiment 5 and the dose-effect relationship of detoxification according to the morphine-dependent mice naloxone, calculating the clinical effective dose scope is 0.22-0.88 μ g/kg/day.
The preparation of other example of formulations has similar result among the present invention in this test.
2, morphine relies on the rat naloxone and urges the withdrawal and treatment test
The Wistar rat is provided by Military Medical Science Institute's animal center, meets national SPF level laboratory animal standard.Body weight 180～220g.Raise in cages 5～6 in every cage.20～24 ℃ of room temperatures, natural lighting, drinking water is not limit.Medicine and reagent: be subjected to the tetraodotoxin lyophilized injectable powder of reagent thing-example of formulations 5, lot number: 070906, provide by Shanghai YiNian Biology Science Co., Ltd.10 μ g/ml are made into desired concn with solvent; Srm-Rhotaard (morphinehydrochloride) is available from Qinghai Pharmaceutic Plant, lot number: 981101; Naloxone hydrochloride: Sigma company produces, and lot number: 42k1184 prepares with physiological saline.Route of administration and volume: subcutaneous injection, administration volume: 0.1ml/10g.Test method: mouse is divided into 5 groups at random, and 12 every group, half and half, 5 group of tetraodotoxin lyophilized injectable powder group that is respectively solvent control group, 4 example of formulations 5 of ♀ ♂.Adopt incremental dose method to set up morphine and rely on rat model.The subcutaneous injection morphine, and every day 2 times (8:00,16:00), 6d continuously.Morphine per daily dose: d 120mg/kg, d 240mg/kg, d 360mg/kg, d 480mg/kg, d 5100mg/kg, d 68:00AM, subcutaneous injection morphine 50mg/kg, behind the 6h, tetraodotoxin lyophilized injectable powder 1,2,4, the 8 μ g/kg of subcutaneous injection example of formulations 5.Behind the 30min, abdominal injection naloxone 2mg/kg puts into the transparent organic glass box of 12cm * 29cm * 15cm (length * wide * height) immediately and observes and Withrawal symptom scoring of record 15min rat and the body weight change of urging front and back 30min and 60min rat.And be base value with the body weight of urgency before giving up, calculate the body weight change rate after urging.Calculation formula: body weight * 100% before body weight change rate (%)=(body weight before the body weight-urgency after the urgency)/urgency.The Withrawal symptom standards of grading of rat: jump (1), wet dog sample shakes body, turn round body, shake the head, yawn, wind up, excitation (being as the criterion to scream): 0 minute=do not have; 1 minute=1-5 time; 2 minutes=6-10 time; 3 minutes＞10 times.(2) tooth quivers, chews (inferior and time between 3s at least at interval): 0 minute=do not have; 1 minute=1-10 time; 2 minutes=11-20 time; 3 minutes＞20 times.(3) hydrostomia, shed tears, hair is upright, blepharoptosis, diarrhoea: 0 minute=do not have; 1 minute=slight; 2 minutes=moderate; 3 minutes=severe.Experimental result is represented with mean ± standard error, adopts the SPSS statistical software to carry out variance analysis (ANOVA), relatively selects the LSD method between group for use.
Table 9. product tetrodotoxin formulation of the present invention relies on the influence of rat withdrawal symptom to morphine
Annotate: mean ± standard error .*P＜0.05, * * P＜0.01, compare with solvent (0 μ g/kg) control group * * * P＜0.001.
On the experiment basis of table 8, further set up morphine and relied on rat model.The experimental result of table 5 shows: after the rat of solvent control group gives naloxone (4mg/kg) urgency, the scoring of Withrawal symptom reaches 12.2 ± 0.91, body weight descended 5.4% and 6.7% respectively after 30min and 60min were given up in urgency, illustrated that administration has formed tangible drug dependence to rat to chronic morphine.Before urgency is given up, the tetraodotoxin lyophilized injectable powder 0.75-6 μ g/kg that gives medicine example of formulations 5 is dose-dependently and reduces the score value that morphine relies on the rat Withrawal symptom, suppress to urge and to give up weight loss behind 30min and the 60min, dosage is 1.5, compare with the solvent control group during 3 and 6 μ g/kg, Withrawal symptom score value and weight loss all obviously alleviate, and show that the tetraodotoxin lyophilized injectable powder of example of formulations 5 has tangible detoxification treatment effect equally to the withdrawal symptom that morphine relies on the urgency of rat naloxone.Rely on the rat naloxone according to morphine and urge the withdrawal and treatment test, the tetraodotoxin freeze-dried powder agent dose of example of formulations 5 and the dose-effect relationship of detoxification, calculating the clinical effective dose scope is 0.23～0.93 μ g/kg/da.
The preparation of other example of formulations has similar result among the present invention in this test.
Claims (5)
1. the preparation method of a tetraodotoxin is characterized in that, described method comprises the steps:
1) ovary of filefish or liver are rubbed, homogenate, stir with the alcoholic solution that contains acetate and to soak, soak solution is filtered, filtrate decompression reclaims alcohol, to there not being the alcohol flavor, crude venom;
2) will go up centrifugal, the filtration of crude venom that the step obtains, get filtrate; Filter residue merges to washings in the filtrate with containing the washing of 3～5% acetic acid water solution, leaves standstill, and separates water layer and upper strata oil reservoir, and oil reservoir is placed and is used for refining Vermiculated puffer liver oil;
3) will go up the water layer in step and filter, filtrate is crossed the weakly acidic cation-exchange resin post, and wash-out is collected elutriant;
4) with the elutriant concentrating under reduced pressure, the concentrated solution of gained is crossed the glucose gel post, and wash-out is collected elutriant, merges concentrating under reduced pressure, gets concentrated solution;
5) use the ammoniacal liquor adjust pH to 8-9 concentrated solution, after placing 45-50 hour under 5～10 ℃ of temperature, sedimentation and filtration, washing, drying, refining, the cut at enrichment TTX peak, concentrating under reduced pressure, lyophilize are promptly.
2. preparation method according to claim 1 is characterized in that, the immersion described in the step 1) is to contain the 30%-100% methyl alcohol of 3～5% acetic acid or ethanolic soln to stir and soak 3～6 times.
3. preparation method according to claim 1 is characterized in that step 2) described in washing for the washing 2-4 time; Leaving standstill under 5-15 ℃ of temperature step 2) placed 8-12 hour.
4. preparation method according to claim 3 is characterized in that step 2) described in washing for the washing 3 times.
5. preparation method according to claim 1 is characterized in that the wash-out in the step 3) is first water wash-out, uses 10% acetic acid wash-out again; Wash-out in the step 4) is for using the 50% ethanolic soln wash-out that contains 5% acetic acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102279942A CN101891751B (en) | 2008-06-18 | 2008-06-18 | Method for preparing tetrodotoxin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102279942A CN101891751B (en) | 2008-06-18 | 2008-06-18 | Method for preparing tetrodotoxin |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200810127045XA Division CN101317846B (en) | 2008-05-28 | 2008-06-18 | Tetrodotoxin formulation for drug rehabilitation , pain ease |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101891751A CN101891751A (en) | 2010-11-24 |
| CN101891751B true CN101891751B (en) | 2011-12-21 |
Family
ID=43101124
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010102279942A Expired - Fee Related CN101891751B (en) | 2008-06-18 | 2008-06-18 | Method for preparing tetrodotoxin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101891751B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104496999A (en) * | 2015-01-06 | 2015-04-08 | 刘学民 | Extraction method of high-purity tetrodotoxin (TTX) |
| CN109157649A (en) * | 2017-06-08 | 2019-01-08 | 赵恩成 | A kind of pharmaceutical applications of scorpion peptide |
| CN107641128B (en) * | 2017-10-09 | 2020-03-17 | 浙江省海洋水产研究所 | Method for efficiently extracting tetrodotoxin |
| CN113321661B (en) * | 2021-05-04 | 2022-04-12 | 中洋生物科技(上海)股份有限公司 | Method for efficiently extracting and separating high-purity tetrodotoxin from globefish viscera |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1425666A (en) * | 2001-12-14 | 2003-06-25 | 上海华腾生物工程有限公司 | Method for extracting tetrodotoxin |
| CN1470515A (en) * | 2003-06-24 | 2004-01-28 | 中国海洋大学 | Method for extracting tetrodotoxin from globefish livers |
| CN1680382A (en) * | 2005-01-19 | 2005-10-12 | 南开大学 | Preparation of tetraodotoxin by two-step resin method and tetraodotoxin preparation thereof |
-
2008
- 2008-06-18 CN CN2010102279942A patent/CN101891751B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1425666A (en) * | 2001-12-14 | 2003-06-25 | 上海华腾生物工程有限公司 | Method for extracting tetrodotoxin |
| CN1470515A (en) * | 2003-06-24 | 2004-01-28 | 中国海洋大学 | Method for extracting tetrodotoxin from globefish livers |
| CN1680382A (en) * | 2005-01-19 | 2005-10-12 | 南开大学 | Preparation of tetraodotoxin by two-step resin method and tetraodotoxin preparation thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101891751A (en) | 2010-11-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| ES2245812T3 (en) | COMPOSITION TO INHIBIT AN INCREASE IN THE BLOOD SUGAR LEVEL OR DECREASE THE BLOOD SUGAR LEVEL. | |
| US20240041984A1 (en) | Methods for producing stable therapeutic formulations in aprotic polar solvents | |
| EA028572B1 (en) | Stable formulation for parenteral injection and method of making and using the same | |
| CN101185692A (en) | Nauclea officinalis extract and preparation and use thereof | |
| CN101317846B (en) | Tetrodotoxin formulation for drug rehabilitation , pain ease | |
| AU2019207531B2 (en) | Peptides and uses thereof | |
| CN101891751B (en) | Method for preparing tetrodotoxin | |
| CN104592025B (en) | Ferulate compound and preparation method thereof | |
| CN100441200C (en) | China fir extracts with lycopodine A and B composition and their preparing method | |
| CN102058593A (en) | Combination medicament of ilaprazole sodium and preparation process thereof | |
| CN1835754A (en) | Stable medical freeze dried preparation of tetraodotoxin | |
| KR20070085519A (en) | An extractive of piper laetispicum c.dc., its process and its uses | |
| KR19980014498A (en) | Rich-sulfur combination preparation | |
| CN1477957A (en) | Tanshinone compound containing dihydrofuran ring structure for curing hyperammonemia and hepatic encephalopathy | |
| JP3334725B2 (en) | Accelerator for reducing alcohol in the body, its metabolites and oral freshener | |
| CN101416936A (en) | Preparation method of long-acting composite sulfamonomethoxine suspension | |
| ES2930757T3 (en) | A drug formulation for use in the effective control of acute and/or chronic pain | |
| CN111789818B (en) | Papaverine hydrochloride pharmaceutical composition for injection and preparation method thereof | |
| CN114028537A (en) | A pharmaceutical composition containing SVHRSP scorpion venom peptide and its preparation method | |
| CN103933100A (en) | Preparation method of total alkaloid of Chinese mahonia stem and applications of total alkaloid of Chinese mahonia stem in preparation of drugs for preventing and treating gastric ulcer | |
| CN1457780A (en) | Total alkaloid composition from plant and its pharmaceutical preparation | |
| CN1320891C (en) | Extractive of Japanese St.Johnswort and preparation method and application | |
| KR100302308B1 (en) | Injection containing high concentrated Aesculus Hippocastanum extract for remedy inflammatory and method of manufacturing the same material | |
| KR100434394B1 (en) | Polyphenol-drug complex using polyphenol extracted from Green tea as a novel drug delivery system and a preparation method thereof | |
| US20180271948A1 (en) | Methods for Producing Stable Therapeutic Glucagon Formulations in Aprotic Polar Solvents |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20111221 Termination date: 20120618 |