CN104984334B - A kind of rabies Attenuate vaccine-praziquantel complexing agent and preparation method and application - Google Patents
A kind of rabies Attenuate vaccine-praziquantel complexing agent and preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of rabies Attenuate vaccine praziquantel complexing agent and preparation method and application, raw material includes praziquantel, double solvent, additives and rabies Attenuate vaccine.The rabies Attenuate vaccine praziquantel complexing agent on the basis of each active ingredient drug effect is ensured, solves the problems, such as that water-soluble rabies Attenuate vaccine is mixed with fat-soluble antiparasitic agent praziquantel stabilization.Preparation method simple possible.
Description
Technical field
The present invention relates to field of medicaments, and in particular to be a kind of rabies Attenuate vaccine-praziquantel complexing agent and its preparation
Method and application.
Background technology
Echinococcosis granulosa (Csystic echinococcusis, CE) also known as echinococcosis (Hydatidosis), are thin
The middle silk ribbon phase parasitized larvae of grain echinococcus (Echinococcus granulosu) in domestic animals such as ox, sheep, pig, camel and horses or
Caused a kind of zoonosis in other organs such as a variety of wild animals or the liver of people, lung.The disease is between humans and animals
It propagates, dog is its definitive host, and sheep, ox are in-between hosts.People can also become in-between host because eating worm's ovum by mistake, and Echinococcus hydatid cyst occurs
Disease.Echinococcosis granulosa seriously endangers the health and life security of human and animal, is current one great public
Hygienic issues.The disease is in worldwide widely distributed, and including Europe, North America, South America, Asia, Africa etc., there are presence in some countries,
The distribution in China is also extremely extensive.This disease is to the growth of infection animal, development, breeding, Animal product quality and quantity and poultry
Animal husbandry development causes to seriously affect.
Echinococcus granulosus middle silk ribbon phase larva --- Echinococcus Granulosus Cysts majority colonizes in the liver of humans and animals (nearly
70%) with lung (nearly 30%), also only a few colonizes in the positions such as heart, spleen, muscle.The harm of Echinococcus Granulosus Cysts is mainly
It excretes poison and the mechanical pressure to internal organs, host can be made to generate inflammatory reaction and allergic reaction.People infects Echinococcus Granulosus Cysts
Afterwards, the symptom occurred due to parasitic site difference is different：In liver, usually cause hepatic region abdominal pain；In lung, often cause cough
It coughs, bloody sputum, pectoralgia；In brain, generally cause the symptoms such as different degrees of nausea and vomiting, headache；It can disability when serious.
After sheep infection Echinococcus Granulosus Cysts, the death rate is higher, and cardinal symptom, which is that hair is inverse, to be stood, loses hair or feathers, collapsing in the ground.Cattle infected particulate spine ball
When the larva of a tapeworm or the cercaria of a schistosome is serious, show as becoming thin, have difficulty in breathing, cough, the output of milk declines etc., the visible chestnut grain of dissect arrives greatly the big particulate spine of football
The ball larva of a tapeworm or the cercaria of a schistosome is encapsulated in liver and/or lung tissue parasitic.The symptom weight of echinococcosis granulosa and the position of Echinococcus Granulosus Cysts parasitism, because
Its tumour size generated, parasitic quantity have very big relationship.It is more universal at present about the prevention and control of echinococcosis granulosa
It is using for a long time to animal antiparasitic agent.
Rabies (Rabies) are also known as hydrophobia, are caused by rabies viruses (Rabies virus), are a kind of holy terrors
Beast is total to illness.Rabic prevention is one of important public health problem in China, has been included in China's communicable disease control systems approach
Surely the second class infectious disease reported.The death rate of the disease is high, once morbidity, the death rate 100%.Up to the present, the world
On still without rabic therapy, antirabic can only occur.The equal easy infection rabies of all warm-blooded animals, most
Susceptible is canid and felid, and canid is rabic major source of infection.With raising dog in society
General mood is prevailing, and not only the quantity of dog greatly increases, but also the chance contacted with people is also more and more, therefore, rabic prevention
Work becomes of crucial importance.The rabic measure of common prevention is that appropriate processing, injection rabies vaccine are carried out to wound and is resisted
Rabic immunoglobulin etc..
As it can be seen that dog is in the propagation and prevalence of rabies and echinococcosis granulosa, it is important and main source.Dog is noted
It penetrates rabies vacciness, take antiparasitic agent, it can effectively both serious Amphixenosis of prevention and control.It is however, common
Antiparasitic agent is fat-soluble, and rabies vacciness is water solubility, and the two can not form homogeneous stabilising system, can only use respectively
Medicine.The difficulty of administration had not only been increased in this way but also has aggravated the working strength of animal doctor, was unfavorable for rabic prevention and parasitic disease
Prevention and control.So find it is a kind of can it is antirabic simultaneously, and can be to such as Echinococcus granulosus, multiceps, blister
The drug that expelling parasite is carried out with the common dog parasite such as tapeworm in tapeworm, diphlidium caninum, line is at present in the anti-of both diseases
The significant problem faced in control.
Invention content
The purpose of the present invention is be directed to technological deficiency in the prior art, on the one hand, provides a kind of simultaneously containing mad
The rabies of rabies vaccine and antiparasitic agent Attenuate vaccine-praziquantel complexing agent is praziquantel, double solvent including main ingredient
Dilution and isometric rabies Attenuate vaccine parenteral solution with additives；It is praziquantel in the dilution, double solvent, additional
Mass volume ratio (the g of agent：mL：ML it is) (6.93:99:1)；Rabies Attenuate vaccine is dense in the rabies Attenuate vaccine parenteral solution
It spends for 1 part/mL.
The double solvent is made of solvent A and solvent B；Solvent A is ethyl lactate；Solvent B is selected from isopropanol, propylene glycol
With one or more in glycerine, preferred isopropanol.
The volume ratio of the solvent A and solvent B is (8：2)-(9:1), preferably 8:2.
The additives are one or more in Tween-80, benzoic acid, salicylic acid, urea, urethane and glycerine,
Preferably Tween-80.
The second aspect of the invention is to provide the method for preparing above-mentioned rabies Attenuate vaccine-praziquantel complexing agent, first
Praziquantel is dissolved in double solvent, is uniformly mixed to obtain dilution with the additives after mixing；It is again that rabies are weak
Malicious seedling is dissolved to obtain rabies Attenuate vaccine parenteral solution by water for injection；Finally by the bodies such as dilution and rabies Attenuate vaccine parenteral solution
Product is uniformly mixed to get rabies Attenuate vaccine-praziquantel complexing agent.
Include the following steps：
(1), praziquantel being placed in autoclaved ampoule bottle, adds in double solvent thereto, sealing, concussion are uniform,
After filtering, praziquantel concentration (quality volumn concentration, g is made：ML) the double solvent praziquantel solution for being 7%；By mad dog
Sick Attenuate vaccine is diluted with water for injection, and the rabies Attenuate vaccine parenteral solution of a concentration of 1 part/mL is made；
(2), double solvent praziquantel solution that step (1) obtains and additives by volume 99 are taken:After 1 mixing, it is placed in
In autoclaved ampoule bottle, sealing, concussion are uniform, and dilution is made；
(3), the rabies Attenuate vaccine parenteral solution that step (1) obtains and the dilution that step (2) obtains is taken to mix in equal volume
Uniformly to get to rabies Attenuate vaccine-praziquantel complexing agent.
The third aspect of the invention, which is to provide, a kind of can allow rabies vacciness and antiparasitic agent to be stable in the presence of together
Dilution in one system, main ingredient include above mentioned praziquantel, double solvent and additives, wherein, praziquantel is answered
Close the mass volume ratio (g of solvent, additives：mL：ML) it is (6.93g:99mL:1mL).
The fourth aspect of the invention is to provide the method for preparing above-mentioned dilution, and praziquantel first is dissolved in double solvent
In, it is uniformly mixed again with additives after mixing, obtains dilution.
The fifth aspect of the invention is that providing above-mentioned rabies Attenuate vaccine-praziquantel complexing agent or the above method prepares
Obtained rabies Attenuate vaccine-praziquantel complexing agent is preparing the application in can preventing the drug of rabies but also expelling parasite.
The expelling parasite refers to drive away common dog parasite, including Echinococcus granulosus, multiceps, blister band tapeworm, dog
Tapeworm etc. in multiple holes tapeworm, line, further includes blood fluke, clonorchis sinensis, lung fluke, fasciolopsis and cysticercus etc..
Rabies Attenuate vaccine-praziquantel complexing agent of the present invention on the basis of each active ingredient drug effect is ensured, solves
The problem that water-soluble rabies Attenuate vaccine is mixed with fat-soluble antiparasitic agent praziquantel stabilization.Its advantage be country to dog into
On the policy foundation of row rabies compulsory immunization, Vaccination and Immunoprophylaxis not only is carried out to rabies, while has also carried out some weights
Infecting both domestic animals and human parasitic disease and canine and domestic animal is wanted to suffer from the prevention and control of parasitic disease altogether, and also reduces the labor in terms of veterinary work
The expenditure of power, material resources and financial resources, produce reality meaning and application future are very great.
Description of the drawings
Fig. 1 show the canonical plotting of mouse rabies virus antibodies standard items；
Fig. 2 show the canonical plotting of praziquantel standard items.
Specific embodiment
Antiparasitic agent (antiparasitic drugs) is primarily referred to as killing, driving away and prevention parasitizes host
The drug of (humans and animals) internal various parasites.Different antiparasitic agents is selected for different parasites.Wherein, pyrrole
Quinoline ketone is the anti-fluke of wide spectrum and tapeworm drug, is a kind of white or off-white color crystalline powder, suitable for various snail fever, China
Clonorchiasis, paragonimiasis, fasciolopsiasis and taeniasis and cysticercosis.Since praziquantel is not soluble in water, also there is first mistake
The shortcomings of effect is big, bioavilability is low, half-life short, therefore be often made into the dosage forms such as liposome, micro-capsule and be administered orally.
The veterinary rabies vaccine of China's production at present mainly includes rabies inactivated vaccine and rabies Attenuate vaccine.Rabies
Attenuate vaccine is a kind of attenuated cell freeze dried vaccine, and for white dry powder-shaped, it is 1 part (use of an animal that specification, which marks one bottle,
Amount), when use directly diluted with water for injection after inject and use.
Clinically, praziquantel drug and rabies vacciness are routinely used alone at present, and praziquantel is taken orally for expelling parasite,
Rabies vacciness injects to prevent rabies.
The present invention wishes to two kinds of drugs compound formulation is made so that single administration to be facilitated to realize multiple prevention and control.Compound formulation
The difficulty faced is that water-soluble rabies Attenuate vaccine is difficult to stablize and mixes with fat-soluble antiparasitic agent praziquantel, is the invention
People has filtered out a kind of double solvent and additives from a variety of solvent systems and cosolvent, can be by praziquantel not soluble in water
It is mixed with the rabies Attenuate vaccine for being dissolved in water, forms stable homogeneous rabies Attenuate vaccine-praziquantel complexing agent.It is administered to animal
Afterwards, under the premise of respective drug effect is ensured, it is antirabic simultaneously, additionally it is possible to carry out expelling parasite to Echinococcus granulosus, one
It lifts two to obtain, improves the efficiency of prevention and control Animal diseases.
Below in conjunction with specific embodiment, present disclosure is further illustrated, and the present invention is further elaborated, but
These embodiments limit the invention absolutely not.
The means used in embodiment, unless otherwise instructed, using the means of this field routine.
Embodiment：Rabies Attenuate vaccine-praziquantel complexing agent and preparation
The rabies Attenuate vaccine that the present embodiment is used be rabies live vaccine (Flury plants), lot number：13021, Rui Pu (protect
It is fixed) Bioceuticals Inc.'s production.Praziquantel is provided by new Katyuan animal pharmaceutical Co. Ltd.
The present invention rabies Attenuate vaccine-praziquantel complexing agent include two kinds of activity of rabies live vaccine and praziquantel into
Point, wherein, dilution need to be first made in praziquantel.Prepare complexing agent the specific steps are：
(1), precise praziquantel 0.07g is placed in autoclaved ampoule bottle, adds in double solvent 1mL thereto
(double solvent is by solvent A and solvent B by volume (8:2)-(9:1) it forms, solvent A is ethyl lactate；Solvent B is selected from isopropyl
It is one or more in alcohol, propylene glycol, glycerine), sealing, concussion are uniform, after 0.22 μm of organic membrane filtration, are made compound molten
Matchmaker's praziquantel solution；
Rabies Attenuate vaccine is diluted to rabies Attenuate vaccine parenteral solution (the weak poison of rabies of 1 part/mL with water for injection
Seedling is existing product, and it is 1 part (dosage of an animal) that specification, which marks one bottle, and when use is directly dilute with 1mL waters for injection
It releases)；
(2), taking double solvent the praziquantel solution 0.99mL and additives 0.01mL that step (1) obtains, (additives are selected from
It is one or more in Tween-80, benzoic acid, salicylic acid, urea, urethane and glycerine) mixing after, be placed in autoclaved peace
In small jar bottle, sealing, concussion are uniform, and dilution is made.
(3), the rabies Attenuate vaccine parenteral solution 1mL that the dilution 1mL that step (2) obtains is obtained with step (1) is taken (by body
Product ratio 1:1) it mixes, rabies Attenuate vaccine-praziquantel complexing agent is made.
As stated above, a series of rabies Attenuate vaccine-praziquantel complexing agents are prepared, the composition of each raw material is shown in Table
1.In production, each raw material dosage can be synchronized by 1 numerical value of table amplifies.
In embodiment listed by table 1, praziquantel dosage is basis《Plumb ' s veterinary drug handbooks》In, praziquantel is disposable to dog
The dosage subcutaneously or intramuscularly injected is about 6mg/kg/ heads, and the weight of a medium dog is generally 10~15kg, therefore praziquantel
Disposable dosage is about 60mg~90mg/ heads.The present embodiment is arranged in pairs or groups 1 using effective injection volume 70mg (0.07g) of praziquantel
Rabies Attenuate vaccine-praziquantel complexing agent is prepared in the rabies Attenuate vaccine of part.
The composition of 1 rabies Attenuate vaccine of table-praziquantel complexing agent
Influence of the double solvent to rabies Attenuate vaccine-praziquantel complexing agent of the present invention of experimental example one, different ratio
In order to study influence of the double solvent of different ratio to rabies Attenuate vaccine-praziquantel complexing agent of the present invention, from
And filter out optimal double solvent, be prepared it is a series of be made of solvent A and solvent B double solvent 1-5 (solvent A with
The volume ratio of solvent B is respectively 7:3、8.5:1.5、9.5:0.5、8:2、8:2), solvent A is ethyl lactate, and solvent B is selected from isopropyl
One or more in alcohol, propylene glycol, glycerine, the volume ratio of solvent A and solvent B adjust and (are shown in Table 2).Make according to the method described above
A series of complexing agent 1-5 (its of rabies Attenuate vaccine-praziquantels is prepared with a series of double solvents that this experimental example obtains
Middle praziquantel is 0.0693g, and rabies Attenuate vaccine (1 part/mL) is 1mL), observe the apparent shape of each complexing agent being prepared
State.Experimental result is shown in Table 2.
The experimental result of table 2 can be seen that when in double solvent the volume ratio of solvent A and solvent B not (8:2)-(9：1)
Range (solvent A accounts for 80-90 volume % in double solvent) it is interior when, manufactured complexing agent can't be homogeneous, clear
Liquid；The too high levels of solvent A, which will appear rabies Attenuate vaccine, can not dissolve and be precipitated in the form of white flock precipitate, solvent
The too high levels of B will appear lamination.And when the volume ratio of solvent A and solvent B is (8:2)-(9：1) it is multiple when in the range of
Mixture is homogeneous clear liquid；And can be the mixing of several substances in solvent B, there is no limit to pyrrole quinoline for mixed proportion
The co-dissolve of ketone and rabies Attenuate vaccine will not have an impact.
Influence of the double solvent of 2 different ratio of table to 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
1st, instrument equipment and reagent：
Multi-function microplate reader Synergy4, the production of Bio-Tek companies of the U.S.；TG16-W high speed centrifugal machine for minim, Changsha Hunan
Instrument centrifuge Instrument Ltd. manufactures；The volley of rifle fire, liquid-transfering gun, German eppendorf manufactures.
Mouse rabies virus antibodies (Rv-Ab) ELISA kit, the abundant Co., Ltd of object all one's life in Shanghai produces.
2nd, experimental animal and grouping：
70 18~22g Kunming, Yunnans system small white mouses are randomly divided into 7 groups.1st group is negative control group, and the 2nd group is sun
Property control group, 3-7 groups be experimental group.Experimental group is according to the body surface area dose lonvestion formula scales of mouse and dog into medicine of equal value
Agent amount.1st group of injecting normal saline is as negative control, and the 2nd group of injection rabies vaccine aqueous solution is as positive control, 3-
Rabies Attenuate vaccine-praziquantel complexing agent of 7 groups of injection embodiment of the present invention 1-5, wherein rabies Attenuate vaccine in 2-7 groups
Content is identical.
3rd, experimental method：
1. mice serum is collected：After mouse normally raises 3-5 days, by the drug to be measured of grouping intramuscular injection 0.5mL, 10d
Afterwards, pluck eyeball of mouse blood sampling 1mL, be placed in centrifuge tube, 3000r/min centrifugation 15min, collect supernatant, be sample to be tested, -20
It DEG C saves backup.
The antibody in sample to be tested is measured according to the specification in mouse rabies virus antibodies (Rv-Ab) ELISA kit
Concentration, standard dilutions therein, enzyme marking reagent, sample diluting liquid, concentrated cleaning solution, color developing agent A, color developing agent B and termination
The reagents such as liquid are all provided by mouse rabies virus antibodies (Rv-Ab) ELISA kit or are provided specific formula, specially：
2. dilution and the sample-adding of mouse rabies virus antibodies standard items：Quasi- 10 holes of sample wells are marked on ELISA Plate,
Add 100 μ L of mouse rabies virus antibodies standard items in one hole, the second hole respectively, standard items are then added in the first hole, the second hole
50 μ L of dilution, mixing；Then 100 μ L are respectively taken from the first hole, the second hole, are added separately to third hole and the 4th hole, then
Add 50 μ L of standard dilutions, mixing in three holes, the 4th hole respectively；In third hole, the 4th hole, 50 μ L is respectively taken to discard, then respectively
50 μ L is taken to be added separately in the 5th hole, the 6th hole, then add 50 μ L of standard dilutions, mixing respectively in the 5th hole, the 6th hole；
50 μ L are respectively taken from the 5th hole, the 6th hole again, are added separately in seven apertures in the human head, octal, then are added respectively in seven apertures in the human head, octal
50 μ L of standard dilutions, mixing；Then 50 μ L are respectively taken from seven apertures in the human head, octal again, are added separately to the 9th hole, the tenth hole
In, then add 50 μ L of standard dilutions, mixing respectively in the 9th hole, the tenth hole；Again 50 μ L are respectively taken from the 9th hole, the tenth hole
It discards.(each hole sample-adding amount is 50 μ L after dilution, and the concentration of mouse rabies virus antibodies standard items is respectively 180pg/mL,
120pg/mL, 60pg/mL, 30pg/mL, 15pg/mL).
3. it is loaded：Blank control wells (being not added with sample to be tested and enzyme marking reagent, remaining each step is identical) and to be measured are set respectively
Sample well.Sample to be tested hole first adds 40 μ L of sample diluting liquid on ELISA Plate, and then adding 10 μ L of sample to be tested again, (sample is finally dilute
Release 5 times), sample to be tested hole repeats to set two.
4. it incubates:With 37 DEG C of incubation 30min after sealing plate film envelope ELISA Plate.
5. with liquid:20 times of concentrated cleaning solutions are released with distilled water 20 again rear spare.
6. it washs:It carefully takes sealing plate film off, discards liquid, dry, cleaning solution is filled it up with per hole, discarded after standing 30s, so
It is repeated 5 times, pats dry.
It is 7. enzyme:50 μ L of enzyme marking reagent are added in per hole, except blank well；It is 4. incubated with step；It is 6. carried out with step
Washing.
8. it develops the color:50 μ L of color developing agent A are added in per hole, 50 μ L of color developing agent B is added, gently vibrates mixing, 37 DEG C are protected from light
Develop the color 15min.
9. it terminates:Add 50 μ L of terminate liquid per hole, terminate reaction.
10. it measures:It is returned to zero with blank well, 450nm wavelength sequentially measures the light absorption value (OD) in each hole, measures and is adding terminate liquid
It is carried out within 15min afterwards.After the absorbance for measuring mouse rabies virus antibodies standard items, using absorbance as ordinate, with mouse
A concentration of abscissa of rabies virus antibodies standard items obtains the standard curve of mouse rabies virus antibodies standard items (such as Fig. 1 institutes
Show) and calibration curve equation (y=0.0115x).The absorbance value of sample to be tested is substituted into calibration curve equation, is obtained to be measured
Mouse rabies virus antibodies concentration in sample, is representative with embodiment 3, remaining embodiment result difference is no different, and is repeated no more,
It the results are shown in Table 3.
3 each group mouse rabies virus antibodies concentration of table
Note：CV (%) coefficient of variation
As can be seen from Table 3, the antibody that embodiment 1-3 groups generate is significantly more than negative control group, with positive controls phase
Than being not significantly different.This just illustrates the mad of the effective content of rabies Attenuate vaccine and positive controls in complexing agent of the present invention
Rabies Attenuate vaccine effective content in dog disease Attenuate vaccine aqueous solution is essentially identical, and complexing agent of the present invention may be used as rabies vaccine
It uses.Experimental result is shown：By resisting to experimental animal injection rabies Attenuate vaccine and rabies Attenuate vaccine-praziquantel complexing agent
The measure of body potency, two groups of generation antibody variabilities are not notable, show praziquantel anthelmintic after being mixed with rabies Attenuate vaccine to mad
The potency of dog disease Attenuate vaccine has no significant effect.
Experimental example three：The assay of praziquantel in praziquantel solution and rabies Attenuate vaccine-praziquantel complexing agent
1st, instrument equipment and reagent：
Electronic balance (BS 224S), the production of Sai Duolisi scientific instrument (Beijing) Co., Ltd；High performance liquid chromatograph
(LC-2010A HT), Japanese Shimadzu Corporation's manufacture；Numerical control ultrasonic cleaner (KQ-700DV), city of Kunshan's ultrasonic instrument are limited
Company produces；Multiplex vavuum pump of circulating water type (SHB- III), Zhengzhou century, available from Shuan Ke laboratory apparatus Co., Ltd.
Praziquantel standard items, lot number：10129462, German Sigma productions；Praziquantel original powder, new Katyuan animal pharmacy have
Limit company provides；It is prepared by Praziquantel injection 7mg/mL, Agricultural University of the Inner Mongol's parasitology laboratory；Methanol, chromatographically pure, moral
State Sigma produces；Rabies Attenuate vaccine be rabies live vaccine (Flury plants), lot number：13021, Rui Pu (Baoding) biology medicine company
Co., Ltd provides.
2nd, experimental method：
1. the foundation of HPLC methods
Chromatographic condition：Chromatographic column：Sigma C18 (250mm × 4.6mm, 5 μm)；Mobile phase：Methanol-water (A/B) gradient is washed
It is de-, 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；Detect wave
It is long：263nm；Column temperature：35℃；Sample size：20μL.
2. the drafting of standard curve：Precision weighs praziquantel standard items, with methanol be configured to a concentration of 0.1mg/mL,
The praziquantel titer of 0.2mg/mL, 0.4mg/mL, 0.6mg/mL, 0.8mg/mL, 1.2mg/mL, 1.6mg/mL.Through 0.45 μm
It after organic membrane filtration, is detected with HPLC, measures the peak area of each concentration praziquantel.Each concentration repeats three times, is obtained
Then the average value of each concentration praziquantel peak area is reduced 106 times, with peak area by the peak area average value of each concentration praziquantel
For ordinate, with a concentration of abscissa of praziquantel, the standard curve of praziquantel peak area-praziquantel concentration is finally drawn out
(see Fig. 2) obtains calibration curve equation (y=1.3639x+0.014).
3. sample recovery rate：Weigh respectively praziquantel standard items be configured to a concentration of 0.84mg/ml, 0.70mg/ml,
The praziquantel standard solution of 0.56mg/ml.The rate of recovery is calculated after HPLC is measured, each concentration is repeated three times, be the results are shown in Table
4。
4. the measure of preci-sion and accuracy：In a few days difference：On the same day interior different time points measured concentration for 0.2mg/mL,
The praziquantel titer of 0.6mg/mL, 1.2mg/mL.Each concentration repeats five times, and calculates the in a few days variation of each concentration samples
Coefficient the results are shown in Table 5.
In the daytime difference：Measured concentration is the praziquantel titer of 0.2mg/ml, 0.6mg/ml, 1.2mg/ml for three days on end.Often
A 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：The praziquantel standard solution of various concentration is prepared with methanol, during with signal-to-noise ratio S/N=3
A concentration of minimum detection limit is minimum quantitative limit during S/N=12.
6. praziquantel parenteral solution assay：After the 100 times of dilutions of methanol of homemade three batches of praziquantel standard solutions
(final concentration is about 0.7mg/mL) through 0.45 μm of organic membrane filtration, under the chromatographic condition of step 1., measures each batch of praziquantel
The peak area of standard solution.Then it by the peak area of praziquantel after 106 times of diminutions are handled, substitutes into calibration curve equation and calculates
The concentration of each batch of praziquantel standard solution is obtained, the results are shown in Table 7.
7. the measure of praziquantel concentration in rabies Attenuate vaccine-praziquantel complexing agent：The complexing agent of embodiment 1-5 is passed through
After 100 times of dilutions (final concentration of praziquantel is about 0.35mg/mL), with 0.45 μm of organic membrane filtration, in the chromatography of step 1.
Under the conditions of, the peak area of praziquantel is measured, finally according to peak area, the concentration of praziquantel in embodiment 1-5 is calculated, with embodiment
3 be representative, remaining embodiment result difference is no different, and is repeated no more, be the results are shown in Table 8.
4 praziquantel sample recovery rate result of table
As can be seen from Table 4, when HPLC methods measure various concentration praziquantel standard solution, sample recovery rate is high, measures
Real result is credible, illustrates that this method can contain for measuring praziquantel in rabies Attenuate vaccine-praziquantel complexing agent of the present invention
Amount.
Difference precision data counts between table 5 days
Difference precision data counts in table 6 days
HPLC methods measure the difference in the daytime and in a few days difference of various concentration praziquantel titer it can be seen from table 5 and table 6
Not significantly, show that this method has higher veracity and precision, can be used in rabies Attenuate vaccine-praziquantel complexing agent
The measure of praziquantel content.
Praziquantel concentration in 7 three batches of praziquantel standard solutions of table
Note：Praziquantel percentage labelled amount：In the recall rate of (0.07) detection praziquantel known to praziquantel content.
Praziquantel concentration in 8 three batches of rabies Attenuate vaccine-praziquantel complexing agents of table
Note：Three reply mixture are Duplicate Samples.
Experimental result is shown (table 7 and table 8) simultaneously：By HPLC methods, praziquantel solution and rabies Attenuate vaccine-pyrrole are measured
As a result the content of praziquantel in quinoline ketone complexing agent is shown in three batches of praziquantel solution and rabies Attenuate vaccine-praziquantel complexing agent
Praziquantel content shows organic solvent and rabies Attenuate vaccine on the content of praziquantel almost without influence more than 90%.
The above is only the preferred embodiment of the present invention, it is noted that for the common skill of the art
For art personnel, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications
Also it should be regarded as protection scope of the present invention.
Claims (11)
1. a kind of rabies Attenuate vaccine-praziquantel complexing agent, which is characterized in that including main ingredient for praziquantel, double solvent
Dilution and isometric rabies Attenuate vaccine parenteral solution with additives；It is praziquantel in the dilution, double solvent, additional
The mass volume ratio of agent is 6.93g:99mL:1mL；A concentration of the 1 of rabies Attenuate vaccine in the rabies Attenuate vaccine parenteral solution
Head part/mL；
The double solvent is made of solvent A and solvent B；Solvent A is ethyl lactate；Solvent B is selected from isopropanol, propylene glycol and sweet
It is one or more in oil；The volume ratio of the solvent A and solvent B is (8：2)-(9:1)；
The additives are one or more in Tween-80, benzoic acid, salicylic acid, urea, urethane and glycerine.
2. rabies Attenuate vaccine-praziquantel complexing agent according to claim 1, which is characterized in that the solvent B is selected from isopropyl
Alcohol.
3. rabies Attenuate vaccine-praziquantel complexing agent according to claim 2, which is characterized in that the solvent A and solvent B
Volume ratio be 8:2.
4. according to any rabies Attenuate vaccine-praziquantel complexing agents of claim 1-3, which is characterized in that the additives
For Tween-80.
5. prepare the method for any rabies Attenuate vaccine-praziquantel complexing agents of claim 1-4, which is characterized in that first will
Praziquantel is dissolved in double solvent, is uniformly mixed to obtain dilution with the additives after mixing；Again by the weak poison of rabies
Seedling is dissolved to obtain rabies Attenuate vaccine parenteral solution by water for injection；It is finally that dilution and rabies Attenuate vaccine parenteral solution is isometric
It is uniformly mixed to get rabies Attenuate vaccine-praziquantel complexing agent.
6. method according to claim 5, which is characterized in that include the following steps：
(1), praziquantel is placed in autoclaved ampoule bottle, adds in double solvent thereto, sealing, concussion are uniform, filtering
Afterwards, praziquantel quality volumn concentration, g is made：ML is 7% double solvent praziquantel solution；Rabies Attenuate vaccine is used
Water for injection dilutes, and the rabies Attenuate vaccine parenteral solution of a concentration of 1 part/mL is made；
(2), double solvent praziquantel solution that step (1) obtains and additives by volume 99 are taken:After 1 mixing, it is placed in high pressure
In the ampoule bottle of sterilizing, sealing, concussion are uniform, and dilution is made；
(3), the rabies Attenuate vaccine parenteral solution that step (1) obtains and the dilution that step (2) obtains is taken to be uniformly mixed in equal volume,
Obtain rabies Attenuate vaccine-praziquantel complexing agent.
7. a kind of dilution in rabies Attenuate vaccine-praziquantel complexing agent, which is characterized in that main ingredient includes right
It is required that 1 to 4 any praziquantel referred to, double solvent and additives, wherein, praziquantel, double solvent, additives mass body
Product is than being 6.93g:99mL:1mL.
8. prepare the method for dilution described in claim 7, which is characterized in that first praziquantel is dissolved in double solvent, is mixed
It is uniformly mixed again with additives after uniformly, obtains dilution.
9. prepared by any rabies Attenuate vaccine-praziquantel complexing agents of claim 1-4 or 5 or 6 the method for claim
Obtained rabies Attenuate vaccine-praziquantel complexing agent is preparing the application in can preventing the drug of rabies but also expelling parasite.
10. apply according to claim 9, which is characterized in that the expelling parasite refers to drive away common dog parasite.
11. apply according to claim 10, which is characterized in that the common dog parasite is Echinococcus granulosus, bull
Tapeworm, blood fluke, clonorchis sinensis, lung fluke, fasciolopsis and/or cysticercus in tapeworm, blister band tapeworm, diphlidium caninum, line.
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