CN104984334A - Hydrophobia attenuated vaccine-praziquantel complex agent and preparation method and application thereof - Google Patents
Hydrophobia attenuated vaccine-praziquantel complex agent and preparation method and application thereof Download PDFInfo
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- CN104984334A CN104984334A CN201510362361.5A CN201510362361A CN104984334A CN 104984334 A CN104984334 A CN 104984334A CN 201510362361 A CN201510362361 A CN 201510362361A CN 104984334 A CN104984334 A CN 104984334A
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Abstract
The invention discloses a hydrophobia attenuated vaccine-praziquantel complex agent and a preparation method and application thereof. The hydrophobia attenuated vaccine-praziquantel complex agent comprises praziquantel, double solvents, additives and hydrophobia attenuated vaccine. According to the hydrophobia attenuated vaccine-praziquantel complex agent, on the basis that the efficacy of all effective constituents is guaranteed, the purpose of stable mixing of the water-soluble hydrophobia attenuated vaccine and the fat-soluble antiparasitic praziquantel is achieved. The preparation method is simple and feasible.
Description
Technical field
The present invention relates to field of medicaments, what be specifically related to is a kind of rabies Attenuate vaccine-praziquantel complexing agent and preparation method thereof and application.
Background technology
Echinococcosis granulosa (Csystic echinococcusis, CE), having another name called echinococcosis (Hydatidosis), is a kind of zoonosis caused in other organs such as liver, lung of the domestic animals such as cattle, sheep, pig, camel and horse or multiple wild animal or people of the middle silk ribbon phase parasitized larvae of Echinococcus granulosus (Echinococcus granulosu).This disease is propagated between humans and animals, and Canis familiaris L. is its final host, and sheep, cattle are its intermediate hosts.People also can become its intermediate host because eating worm's ovum by mistake, and echinococcosis occurs.The healthy of human and animal and life security in echinococcosis granulosa serious harm, are current great public health problems.This disease is worldwide extensive distribution, and comprising some countries such as Europe, North America, South America, Asia, Africa has existence, and the distribution of China is also extremely extensive.This disease all causes the growth of infection animal, growth, breeding, Animal product quality and quantity and animal husbandry development and has a strong impact on.
Middle silk ribbon phase larva of Echinococcus granulosus---Echinococcus Granulosus Cysts majority colonizes in liver (nearly 70%) and the lung (nearly 30%) of humans and animals, and only a few colonizes in the positions such as heart, spleen, muscle in addition.The harm of Echinococcus Granulosus Cysts mainly excretes poison and mechanical pressure to internal organs, and host can be made to produce inflammatory reaction and anaphylaxis.After people infects Echinococcus Granulosus Cysts, the symptom occurred because parasitic site is different is different: at liver, usually cause hepatic region to suffer from abdominal pain; In pulmonary, often cause cough, bloody sputum, chest pain; At brain, generally cause the symptoms such as nausea and vomiting in various degree, headache; Can disability time serious.After sheep infects Echinococcus Granulosus Cysts, mortality rate is higher, and cardinal symptom is that hair is against standing, losing hair or feathers, collapse in the ground.When cattle infected Echinococcus Granulosus Cysts is serious, show as become thin, dyspnea, cough, degradation under milk yield, cut open the visible chestnut grain of inspection and be encapsulated in liver and/or lung tissue parasitic to the Echinococcus Granulosus Cysts that football is large greatly.The symptom weight of echinococcosis granulosa and the position of Echinococcus Granulosus Cysts parasitism, there is very large relation because of its cyst size produced, parasitic quantity.At present about the prevention and control of echinococcosis granulosa, comparatively generally adopt long-term to animal antiparasitic.
Rabies (Rabies), also known as hydrophobia, are caused by rabies virus (Rabies virus), are that a kind of holy terror beast is ill altogether.Rabic control is one of important public health problem of China, has included the Equations of The Second Kind infectious disease of China's communicable disease control system statutory report in.The mortality rate of this disease is high, once morbidity, mortality rate is 100%.Up to the present, still there is no rabic Therapeutic Method in the world, can only antirabicly occur.The equal easy infection rabies of all homoiothermic animals, most susceptible be Canis animals and felid, and Canis animals is rabic major source of infection.Along with the pathogenic wind of society raising Canis familiaris L. is prevailing, not only the quantity of Canis familiaris L. increases greatly, and the chance contacted with people also gets more and more, and therefore, rabic prevention work becomes of crucial importance.The rabic measure of common prevention is carry out suitable process, injection rabies vaccine and antilyssic immunoglobulin etc. to wound.
Visible, dog rabies and echinococcosis granulosa propagation and popular on, be important and main source.To dog injecting pstudorabies vaccine, take antiparasitic, can prevention and control these two kinds are serious effectively Amphixenosis.But conventional antiparasitic is fat-soluble, and rabies vaccine is water solublity, and both cannot form the stabilising system of homogeneous phase, can only distinguish medication.So not only increase the difficulty of administration but also increased the weight of the working strength of veterinary, being unfavorable for the prevention and control of rabic prevention and parasitic disease.Can antirabic while so find one, the medicine that can carry out anthelmintic to common dog parasites such as cestodes in such as Echinococcus granulosus, multiceps, blister band cestode, diphlidium caninum, line is again the significant problem faced in the prevention and control of these two kinds of diseases at present.
Summary of the invention
The object of the invention is the technological deficiency for existing in prior art, on the one hand, a kind of rabies Attenuate vaccine-praziquantel complexing agent simultaneously containing rabies vaccine and antiparasitic is provided, comprises diluent and isopyknic rabies Attenuate vaccine injection that Main Ingredients and Appearance is praziquantel, double solvent and additives; In described diluent, the mass volume ratio (g:mL:mL) of praziquantel, double solvent, additives is (6.93:99:1); In described rabies Attenuate vaccine injection, the concentration of rabies Attenuate vaccine is 1 part/mL.
Described double solvent is made up of solvent A and solvent B; Solvent A is ethyl lactate; Solvent B be selected from isopropyl alcohol, propylene glycol and glycerol one or more, preferred isopropyl alcohol.
The volume ratio of described solvent A and solvent B is (8:2)-(9:1), is preferably 8:2.
Described additives be selected from tween 80, benzoic acid, salicylic acid, carbamide, urethane and glycerol one or more, be preferably tween 80.
Second aspect of the present invention is to provide the method preparing above-mentioned rabies Attenuate vaccine-praziquantel complexing agent, is first dissolved in double solvent by praziquantel, mixs homogeneously obtain diluent after mix homogeneously with described additives; Again rabies Attenuate vaccine is dissolved by water for injection and obtain rabies Attenuate vaccine injection; Finally diluent is mixed homogeneously with rabies Attenuate vaccine injection equal-volume, obtain rabies Attenuate vaccine-praziquantel complexing agent.
Comprise the following steps:
(1), by praziquantel be placed in autoclaved ampoule bottle, add double solvent wherein, sealing, concussion evenly, after filtration, make the double solvent praziquantel solution that praziquantel concentration (quality volumn concentration, g:mL) is 7%; Rabies Attenuate vaccine water for injection is diluted, makes the rabies Attenuate vaccine injection that concentration is 1 part/mL;
(2), get double solvent praziquantel solution that step (1) obtains 99:1 mix by volume with additives after, be placed in autoclaved ampoule bottle, sealing, concussion evenly, make diluent;
(3), get rabies Attenuate vaccine injection that step (1) obtains and the diluent equal-volume mix homogeneously that step (2) obtains, namely obtain rabies Attenuate vaccine-praziquantel complexing agent.
3rd aspect of the present invention is to provide a kind of rabies vaccine and the diluent of antiparasitic stable existence in same system of allowing, Main Ingredients and Appearance comprises above mentioned praziquantel, double solvent and additives, wherein, the mass volume ratio (g:mL:mL) of praziquantel, double solvent, additives is (6.93g:99mL:1mL).
4th aspect of the present invention is to provide the method preparing above-mentioned diluent, is first dissolved in double solvent by praziquantel, mixs homogeneously after mix homogeneously with additives again, obtain diluent.
Rabies Attenuate vaccine-praziquantel complexing agent that 5th aspect of the present invention is to provide above-mentioned rabies Attenuate vaccine-praziquantel complexing agent or said method to prepare preparation can preventing rabies again can application in the medicine of anthelmintic.
Described anthelmintic refers to drives away common dog parasite, to comprise in Echinococcus granulosus, multiceps, blister band cestode, diphlidium caninum, line cestode etc., also comprises schistosomicide, clonorchis sinensis, lung fluke, fasciloopsis and cysticercosis etc.
Rabies Attenuate vaccine-praziquantel complexing agent of the present invention, on the basis ensureing each effective ingredient drug effect, solves water solublity rabies Attenuate vaccine and stablizes with fat-soluble antiparasitic praziquantel the difficult problem mixed.Its advantage is carried out on the policy foundation of rabies compulsory immunization in country to dog, not only Vaccination and Immunoprophylaxis is carried out to rabies, also the prevention and control that some important infecting both domestic animals and human parasitic disease and dog class and domestic animal suffer from parasitic disease have altogether been carried out simultaneously, and decreasing the expenditure of the labour of veterinary work aspect, material resources and financial resources, it produces practical significance and application future is very great.
Accompanying drawing explanation
Figure 1 shows that the canonical plotting of mice rabies virus antibodies standard substance;
Figure 2 shows that the canonical plotting of praziquantel standard substance.
Detailed description of the invention
Antiparasitic (antiparasitic drugs) mainly refers to for killing, driving away and prevention parasitizes various parasitic medicine in host's (humans and animals) body.Different antiparasitics is selected for different parasites.Wherein, praziquantel is the anti-trematodiasis of wide spectrum and cestode medicine, is a kind of white or off-white color crystalline powder, is applicable to various schistosomicide, clonorchiasis sinensis, paragonimiasis westermani, fasciolopsiasis buski and taeniasis and cysticercosis.Because praziquantel is water insoluble, also there is the shortcomings such as first pass effect is large, bioavailability is low, the half-life is short, be therefore often made into the dosage form such as liposome, microcapsule and orally used.
The veterinary rabies vaccine that current China produces mainly comprises rabies inactivated vaccine and rabies Attenuate vaccine.Rabies Attenuate vaccine is a kind of attenuated cell freeze dried vaccine, is white powdered, and it is 1 part (consumption of an animal) that description marks one bottle, directly injects use with after water for injection dilution during use.
Clinically, current praziquantel medicine and rabies vaccine are used alone all routinely, and praziquantel is oral for anthelmintic, and rabies vaccine injection is used for preventing rabies.
The present invention wishes that two kinds of medicines can be made compound formulation realizes multiple prevention and control to facilitate single administration.The difficulty that compound formulation faces is that water solublity rabies Attenuate vaccine is difficult to stable mixing with fat-soluble antiparasitic praziquantel, inventor has filtered out a kind of double solvent and additives from multiple solvent system and cosolvent for this reason, by water-fast praziquantel and the mixing of water-soluble rabies Attenuate vaccine, stable homogeneous phase rabies Attenuate vaccine-praziquantel complexing agent can be formed.After being administered to animal, under the prerequisite ensureing respective drug effect, antirabic while, anthelmintic can also be carried out to Echinococcus granulosus, kill two birds with one stone, improve the efficiency of prevention and control Animal diseases.
Below in conjunction with specific embodiment, further illustrate content of the present invention, and the present invention is further elaborated, but these embodiments limit the invention absolutely not.
The means used in embodiment, if no special instructions, all use the means of this area routine.
Embodiment: rabies Attenuate vaccine-praziquantel complexing agent and preparation
The rabies Attenuate vaccine that the present embodiment is used is rabies live vaccine (Flury strain), lot number: 13021, and Ruipu (Baoding) Biological Pharmaceutical Co., Ltd. produces.Praziquantel is provided by new Katyuan animal pharmaceutical Co. Ltd.
Rabies Attenuate vaccine-praziquantel complexing agent of the present invention comprises rabies live vaccine and praziquantel two kinds of active component, and wherein, praziquantel first need make diluent.The concrete steps preparing complexing agent are:
(1), precise praziquantel 0.07g, be placed in autoclaved ampoule bottle, add wherein double solvent 1mL (double solvent by solvent A and solvent B by volume (8:2)-(9:1) form, solvent A is ethyl lactate; Solvent B be selected from isopropyl alcohol, propylene glycol, glycerol one or more), sealing, concussion evenly, after 0.22 μm of organic membrane filtration, make double solvent praziquantel solution;
(rabies Attenuate vaccine is existing product rabies Attenuate vaccine water for injection to be diluted to the rabies Attenuate vaccine injection of 1 part/mL, it is 1 part (consumption of an animal) that description marks one bottle, directly dilutes with 1mL water for injection during use);
(2), get after double solvent praziquantel solution 0.99mL that step (1) obtains and additives 0.01mL (additives be selected from tween 80, benzoic acid, salicylic acid, carbamide, urethane and glycerol one or more) mix, be placed in autoclaved ampoule bottle, sealing, concussion evenly, make diluent.
(3), get the diluent 1mL that step (2) obtains and mix with rabies Attenuate vaccine injection 1mL (by volume 1:1) that step (1) obtains, make rabies Attenuate vaccine-praziquantel complexing agent.
As stated above, prepare a series of rabies Attenuate vaccine-praziquantel complexing agent, the composition of its each raw material is in table 1.In production, each raw material dosage synchronously can be amplified by table 1 numerical value.
In embodiment listed by table 1, praziquantel consumption is according in " Plumb ' s veterinary drug handbook ", praziquantel is about 6mg/kg/ head to the disposable consumption that is subcutaneous or intramuscular injection of dog, and the body weight of a medium dog is generally 10 ~ 15kg, and therefore the disposable consumption of praziquantel is about 60mg ~ 90mg/ head.The present embodiment adopts the arrange in pairs or groups rabies Attenuate vaccine of 1 part of effective injection volume 70mg (0.07g) of praziquantel to prepare rabies Attenuate vaccine-praziquantel complexing agent.
The composition of table 1 rabies Attenuate vaccine-praziquantel complexing agent
The double solvent of experimental example one, different ratio is on the impact of rabies Attenuate vaccine-praziquantel complexing agent of the present invention
In order to study the double solvent of different ratio to the impact of rabies Attenuate vaccine-praziquantel complexing agent of the present invention, thus filter out optimum double solvent, prepare a series of double solvent 1-5 (volume ratio of solvent A and solvent B is respectively 7:3,8.5:1.5,9.5:0.5,8:2,8:2) be made up of solvent A and solvent B, solvent A is ethyl lactate, solvent B be selected from isopropyl alcohol, propylene glycol, glycerol one or more, the volume ratio of solvent A and solvent B adjusts (see table 2).The a series of double solvents using this experimental example to obtain according to the method described above prepare the complexing agent 1-5 of a series of rabies Attenuate vaccine-praziquantel, and (wherein praziquantel is 0.0693g, rabies Attenuate vaccine (1 part/mL) is 1mL), observe the apparent state of each complexing agent prepared.Experimental result is in table 2.
The experimental result of table 2 can be found out, when the volume ratio of solvent A and solvent B in double solvent is not in the scope of (8:2)-(9:1) (solvent A accounts for 80-90 volume % in double solvent), the complexing agent made all can not become the liquid of homogeneous phase, clarification; The too high levels of solvent A there will be rabies Attenuate vaccine and cannot dissolve and separate out with the form of white flock precipitate, and the too high levels of solvent B there will be lamination.And when the volume ratio of solvent A and solvent B is in the scope of (8:2)-(9:1), complexing agent is the liquid of homogeneous phase clarification; And can be the mixing of several material in solvent B, mixed proportion limit, and can not have an impact to the common dissolving of praziquantel and rabies Attenuate vaccine.
The double solvent of table 2 different ratio is on the impact of rabies Attenuate vaccine-praziquantel complexing agent of the present invention
Experimental example two: the antibody concentration of rabies Attenuate vaccine-praziquantel complexing agent of the present invention measures
1, instrument equipment and reagent:
Multi-functional microplate reader Synergy4, Bio-Tek company of the U.S. produces; TG16-W high speed centrifugal machine for minim, Changsha Xiang Yi centrifuge Instrument Ltd. manufactures; The volley of rifle fire, liquid-transfering gun, German eppendorf manufactures.
Mice rabies virus antibodies (Rv-Ab) ELISA kit, Shanghai abundant thing all one's life company limited produces.
2, laboratory animal and grouping:
70 18 ~ 22g Kunming, Yunnan system white mice are divided into 7 groups at random.1st group is negative control group, and the 2nd group is positive controls, and 3-7 group is experimental group.Experimental group becomes drug dose of equal value according to mice with the body surface area dose lonvestion formula scales of dog.1st group of injecting normal saline is as negative control, and the 2nd group of injection rabies vaccine aqueous solution is as positive control, and the rabies Attenuate vaccine-praziquantel complexing agent of 3-7 group injection embodiment of the present invention 1-5, wherein in 2-7 group, the content of rabies Attenuate vaccine is identical.
3, experimental technique:
1. mice serum is collected: after mice normally raises 3-5 days, and by the medicine to be measured of grouping intramuscular injection 0.5mL, after 10d, pluck eyeball of mouse blood sampling 1mL, be placed in centrifuge tube, the centrifugal 15min of 3000r/min, collect supernatant, be testing sample ,-20 DEG C save backup.
The antibody concentration in testing sample is measured according to the description in mice rabies virus antibodies (Rv-Ab) ELISA kit, the reagent such as standard dilutions wherein, enzyme marking reagent, sample diluting liquid, concentrated cleaning solution, developer A, developer B and stop buffer all provide or provide concrete formula by mice rabies virus antibodies (Rv-Ab) ELISA kit, are specially:
2. the dilution of mice rabies virus antibodies standard substance and application of sample: the accurate sample wells of bidding 10 holes in ELISA Plate, mice rabies virus antibodies standard substance 100 μ L is added respectively in the first hole, the second hole, then in the first hole, the second hole, standard dilutions 50 μ L is added, mixing; Then from the first hole, the second hole, respectively get 100 μ L, be added to the 3rd hole and the 4th hole respectively, then add standard dilutions 50 μ L respectively in the 3rd hole, the 4th hole, mixing; In the 3rd hole, the 4th hole, respectively get 50 μ L and discard, respectively get 50 μ L and be added to respectively in the 5th hole, the 6th hole, then add standard dilutions 50 μ L respectively in the 5th hole, the 6th hole, mixing; From the 5th hole, the 6th hole, respectively get 50 μ L again, be added in seven apertures in the human head, octal respectively, then in seven apertures in the human head, octal, add standard dilutions 50 μ L respectively, mixing; And then 50 μ L are respectively got from seven apertures in the human head, octal, be added to respectively in the 9th hole, the tenth hole, then add standard dilutions 50 μ L respectively in the 9th hole, the tenth hole, mixing; From the 9th hole, the tenth hole, respectively get 50 μ L again to discard.(after dilution, each hole application of sample amount is 50 μ L, and the concentration of mice rabies virus antibodies standard substance is respectively 180pg/mL, 120pg/mL, 60pg/mL, 30pg/mL, 15pg/mL).
3. application of sample: establish blank control wells (do not add testing sample and enzyme marking reagent, all the other each steps are identical) and testing sample hole respectively.In ELISA Plate, testing sample hole first adds sample diluting liquid 40 μ L, and then adds testing sample 10 μ L (sample finally dilutes 5 times), and testing sample hole repeats to establish two.
4. incubation: with 37 DEG C of incubation 30min after shrouding film envelope ELISA Plate.
5. dosing: 20 times of concentrated cleaning solution distilled water 20 are doubly released rear for subsequent use.
6. wash: carefully take shrouding film off, discard liquid, dry, cleaning mixture is filled it up with in every hole, discards after leaving standstill 30s, so repeats 5 times, pats dry.
7. enzyme-added: every hole adds enzyme marking reagent 50 μ L, except blank well; 4. incubation is carried out with step; 6. wash with step.
8. develop the color: every hole adds developer A 50 μ L, then adds developer B 50 μ L, mixing of vibrating gently, 37 DEG C of lucifuge colour developing 15min.
9. stop: every hole adds stop buffer 50 μ L, cessation reaction.
10. measure: with blank well zeroing, 450nm wavelength sequentially measures the light absorption value (OD) in each hole, is determined at after adding stop buffer and carries out within 15min.After recording the absorbance of mice rabies virus antibodies standard substance, take absorbance as vertical coordinate, with the concentration of mice rabies virus antibodies standard substance for abscissa, obtain standard curve (as shown in Figure 1) and the standard curve equation (y=0.0115x) of mice rabies virus antibodies standard substance.Substituted in standard curve equation by the absorbance of testing sample, obtain the mice rabies virus antibodies concentration in testing sample, with embodiment 3 for representative, all the other embodiment results difference is as good as, and repeats no more, the results are shown in Table 3.
Table 3 is group mice rabies virus antibodies concentration respectively
Note: CV (%) coefficient of variation
As can be seen from Table 3, the antibody that embodiment 1-3 group produces is obviously many than negative control group, compared with positive controls, does not have significant difference.This is substantially identical with regard to the rabies Attenuate vaccine effective content in the effective content of rabies Attenuate vaccine in explanation complexing agent of the present invention and the rabies Attenuate vaccine aqueous solution of positive controls, and complexing agent of the present invention can be used as rabies vaccine and use.Experimental result shows: by the mensuration to laboratory animal injection rabies Attenuate vaccine and rabies Attenuate vaccine-praziquantel complexing agent antibody titer, two groups produce antibody variability significantly, show that praziquantel anthelmintic has no significant effect tiring of rabies Attenuate vaccine after mixing with rabies Attenuate vaccine.
Experimental example three: the assay of praziquantel in praziquantel solution and rabies Attenuate vaccine-praziquantel complexing agent
1, instrument equipment and reagent:
Electronic balance (BS 224S), Sai Duolisi scientific instrument (Beijing) company limited produces; High performance liquid chromatograph (LC-2010A HT), Japanese Shimadzu Corporation manufactures; Numerical control ultrasonic cleaner (KQ-700DV), Kunshan Ultrasonic Instruments Co., Ltd. produces; The multiplex vacuum pump of circulating water type (SHB-III), Zhengzhou century, available from Shuan Ke experimental apparatus company limited.
Praziquantel standard substance, lot number: 10129462, German Sigma produces; The former powder of praziquantel, new Katyuan animal pharmaceutical Co. Ltd provides; Praziquantel injection 7mg/mL, Agricultural University of the Inner Mongol's parasitology laboratory preparation; Methanol, chromatographically pure, German Sigma produces; Rabies Attenuate vaccine is rabies live vaccine (Flury strain), and lot number: 13021, Ruipu (Baoding) Biological Pharmaceutical Co., Ltd. provides.
2, experimental technique:
1. the foundation of HPLC method
Chromatographic condition: chromatographic column: sigma C18 (250mm × 4.6mm, 5 μm); Mobile phase: methanol-water (A/B) gradient elution, i.e. 0.01min-2.00min, A/B=60/40; 2.00min-10min, A/B=60/40-68/32; 10.00-12.00min, A/B=68/32-60/40; 12.00min-15.00min, A/B=60/40; Flow velocity 1.0mL/min; Determined wavelength: 263nm; Column temperature: 35 DEG C; Sample size: 20 μ L.
2. the drafting of standard curve: precision takes praziquantel standard substance, is mixed with methanol the praziquantel titer that concentration is 0.1mg/mL, 0.2mg/mL, 0.4mg/mL, 0.6mg/mL, 0.8mg/mL, 1.2mg/mL, 1.6mg/mL.After 0.45 μm of organic membrane filtration, detect with HPLC, measure the peak area of each concentration praziquantel.Each concentration repeats three times, obtain the peak area meansigma methods of each concentration praziquantel, then the meansigma methods of each concentration praziquantel peak area is reduced 106 times, take peak area as vertical coordinate, with the concentration of praziquantel for abscissa, finally draw out the standard curve (see Fig. 2) of praziquantel peak area-praziquantel concentration, obtain standard curve equation (y=1.3639x+0.014).
3. average recovery: take praziquantel standard substance respectively and be mixed with the praziquantel standard solution that concentration is 0.84mg/ml, 0.70mg/ml, 0.56mg/ml.After HPLC measures, calculate the response rate, each concentration repeats three times, the results are shown in Table 4.
4. the mensuration of preci-sion and accuracy: in a few days difference: on the same day, different time points measures the praziquantel titer that concentration is 0.2mg/mL, 0.6mg/mL, 1.2mg/mL.Each concentration repeats five times, calculates the in a few days coefficient of variation of each concentration samples, the results are shown in Table 5.
In the daytime difference: measure the praziquantel titer that concentration is 0.2mg/ml, 0.6mg/ml, 1.2mg/ml for three days on end.Each concentration repeats three times, calculates the coefficient of variation in the daytime of each concentration samples, the results are shown in Table 6.
5. sensitivity is investigated: with the praziquantel standard solution of methanol preparation variable concentrations, with concentration during signal to noise ratio S/N=3 for lowest detectable limit, be minimum quantitative limit during S/N=12.
6. praziquantel injection assay: by after homemade three batches of praziquantel standard solution methanol 100 times dilution (final concentration is about 0.7mg/mL), through 0.45 μm of organic membrane filtration, under step chromatographic condition 1., measure the peak area of each batch of praziquantel standard solution.Then, after the peak area of praziquantel being reduced process through 106 times, substitute into the concentration that standard curve Equation for Calculating obtains each batch of praziquantel standard solution, the results are shown in Table 7.
7. the mensuration of praziquantel concentration in rabies Attenuate vaccine-praziquantel complexing agent: by the complexing agent of embodiment 1-5 after 100 times of dilutions (final concentration of praziquantel is about 0.35mg/mL), with 0.45 μm of organic membrane filtration, under step chromatographic condition 1., measure the peak area of praziquantel, finally according to peak area, calculate the concentration of praziquantel in embodiment 1-5, with embodiment 3 for representative, all the other embodiment results difference is as good as, and repeats no more, the results are shown in Table 8.
Table 4 praziquantel average recovery result
As can be seen from Table 4, when HPLC method measures variable concentrations praziquantel standard solution, average recovery is high, and measurement result is genuine and believable, illustrates that the method can be used for measuring praziquantel content in rabies Attenuate vaccine-praziquantel complexing agent of the present invention.
Difference precision data statistics between table 5 day
Difference precision data statistics in table 6 day
As can be seen from table 5 and table 6, HPLC method measures the difference in the daytime of variable concentrations praziquantel titer and in a few days difference is not remarkable, show that the method has higher veracity and precision, may be used for the mensuration of praziquantel content in rabies Attenuate vaccine-praziquantel complexing agent.
Praziquantel concentration in table 7 three batches of praziquantel standard solutions
Note: praziquantel percentage labelled amount: (0.07) detects the recall rate of praziquantel when praziquantel content is known.
Praziquantel concentration in table 8 three batches of rabies Attenuate vaccine-praziquantel complexing agents
Note: three reply mixture are Duplicate Samples.
Experimental result shows simultaneously (table 7 and table 8): by HPLC method, measure the content of praziquantel in praziquantel solution and rabies Attenuate vaccine-praziquantel complexing agent, result shows praziquantel content in three batches of praziquantel solution and rabies Attenuate vaccine-praziquantel complexing agent and, all more than 90%, shows organic solvent and rabies Attenuate vaccine to the content of praziquantel almost without impact.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. rabies Attenuate vaccine-praziquantel complexing agent, is characterized in that, comprises diluent and isopyknic rabies Attenuate vaccine injection that Main Ingredients and Appearance is praziquantel, double solvent and additives; In described diluent, the mass volume ratio (g:mL:mL) of praziquantel, double solvent, additives is (6.93:99:1); In described rabies Attenuate vaccine injection, the concentration of rabies Attenuate vaccine is 1 part/mL.
2. rabies Attenuate vaccine-praziquantel complexing agent according to claim 1, it is characterized in that, described double solvent is made up of solvent A and solvent B; Solvent A is ethyl lactate; Solvent B be selected from isopropyl alcohol, propylene glycol and glycerol one or more, preferred isopropyl alcohol.
3. rabies Attenuate vaccine-praziquantel complexing agent according to claim 2, it is characterized in that, the volume ratio of described solvent A and solvent B is (8:2)-(9:1), is preferably 8:2.
4., according to the arbitrary described rabies Attenuate vaccine-praziquantel complexing agent of claim 1-3, it is characterized in that, described additives be selected from tween 80, benzoic acid, salicylic acid, carbamide, urethane and glycerol one or more, be preferably tween 80.
5. prepare the method for the arbitrary described rabies Attenuate vaccine-praziquantel complexing agent of claim 1-4, it is characterized in that, first praziquantel is dissolved in double solvent, mix homogeneously with described additives after mix homogeneously and obtain diluent; Again rabies Attenuate vaccine is dissolved by water for injection and obtain rabies Attenuate vaccine injection; Finally diluent is mixed homogeneously with rabies Attenuate vaccine injection equal-volume, obtain rabies Attenuate vaccine-praziquantel complexing agent.
6. method according to claim 5, is characterized in that, comprise the following steps:
(1), by praziquantel be placed in autoclaved ampoule bottle, add double solvent wherein, sealing, concussion evenly, after filtration, make the double solvent praziquantel solution that praziquantel concentration (quality volumn concentration, g:mL) is 7%; Rabies Attenuate vaccine water for injection is diluted, makes the rabies Attenuate vaccine injection that concentration is 1 part/mL;
(2), get double solvent praziquantel solution that step (1) obtains 99:1 mix by volume with additives after, be placed in autoclaved ampoule bottle, sealing, concussion evenly, make diluent;
(3), get rabies Attenuate vaccine injection that step (1) obtains and the diluent equal-volume mix homogeneously that step (2) obtains, namely obtain rabies Attenuate vaccine-praziquantel complexing agent.
7. one kind is used in the diluent in rabies Attenuate vaccine-praziquantel complexing agent, it is characterized in that, Main Ingredients and Appearance comprises arbitrary praziquantel, double solvent and the additives mentioned of Claims 1-4, wherein, the mass volume ratio (g:mL:mL) of praziquantel, double solvent, additives is (6.93g:99mL:1mL).
8. prepare the method for diluent described in claim 7, it is characterized in that, first praziquantel is dissolved in double solvent, mixs homogeneously with additives again after mix homogeneously, obtain diluent.
9. rabies Attenuate vaccine-praziquantel complexing agent of preparing of method described in the arbitrary described rabies Attenuate vaccine-praziquantel complexing agent of claim 1-4 or claim 5 or 6 preparation can preventing rabies again can application in the medicine of anthelmintic.
10. apply according to claim 9, it is characterized in that, described anthelmintic refers to drives away common dog parasite, to comprise in Echinococcus granulosus, multiceps, blister band cestode, diphlidium caninum, line cestode etc., also comprises schistosomicide, clonorchis sinensis, lung fluke, fasciloopsis and cysticercosis etc.
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