CN104055780B - Rat compound anesthetic and preparation method thereof - Google Patents
Rat compound anesthetic and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of rat compound anesthetic and preparation method thereof, belong to preparation and the application of animal compound anesthetic.First the present invention discloses a kind of rat compound anesthetic, including: dexmedetomidine, midazolam, oxycodone, buffer and water for injection.The invention also discloses the preparation method of a kind of rat animal compound anesthetic, above-mentioned each composition be dissolved in water for injection, dilution, filter, embedding in ampoule bottle, sterilizing, to obtain final product.Rat disclosed by the invention is rapid with compound anesthetic induction of anesthesia, and the anesthesia maintenance time is long, and analgesia, sedation effect are good, revive steadily, to the major physiological biochemical of rat all in the range of its physiological tolerance, simple to operate, can be used for that rat is complicated or the operation of time length.
Description
Technical field
The present invention relates to a kind of compound anesthetic, particularly relate to a kind of rat compound anesthetic and system thereof
Preparation Method, belongs to preparation and the application of murine compound anesthetic.
Background technology
In zoopery, selecting anesthesia suitable, safe and effective is experiment safety and number
Important foundation according to reliability.Rat is usually used in medicine Nutrition and Metabolism, endocrine, behavioristics, old age
The research of the aspects such as sick, tumor, infectious disease, cardiovascular disease and the preparation of surgery models,
And the improper a series of pathophysiological change that may cause murine body of anesthesia, even cause
It is dead, affects the result of whole experiment.
Current domestic conventional rat anesthesia preparation has chloral hydrate, urethane, pentobarbital, chloramines
Ketone, Su Mian Xin etc..Chloral hydrate is short acting anesthetic, and the comparatively safe scope of 10% chloral hydrate is relatively
Little, depth of anesthesia is shallower, of flaccid muscles and analgesic effect is the best, fundamentally cannot be used complicated and time-consuming
In longer operation process, when being used alone, mortality rate is of a relatively high, and often needs boost.Crow
La Tan is Lente anesthetic agent, when depth of anesthesia reaches surgical operation requirement, to breathing and circulate without substantially
Inhibitory action, but this medicine toxicity is big, there are some researches show that 20% urethane anesthesia can make Rat Erythrocytes
Aggregation increases, therefore should not be used in hemorheological related experiment, has proven to urethane to rat
Blood has a significant impact, and itself has carcinogenecity, therefore does not recommends in the general anesthesia of laboratory animal
Such anesthetics.Pentobarbital sodium anesthesia maintenance time about 2h, but various to visual electrophysiology
Waveform influence is relatively big, and lumbar injection has certain pressing down to animal body cardiovascular, nervous system simultaneously
Making use, body damage is bigger.Ketamine is short acting anesthetic, 120mg/kg lumbar injection rat,
Anesthesia maintenance time only about 45min, narrow application range.Although Su Mian Xin can be by controlling dosage
The regulation anesthesia maintenance time, but bigger to the b wave action of rat retina electrograph in anaesthesia process.
Although the inhalation anesthesia of rat achieves effectively control on depth of anesthesia, but needs when using
By special anesthesia equipment, it is equipped with single-minded inhalation anesthesia Volatile anesthetic.Not only formality is the most loaded down with trivial details, and
And equipment and medicine sufficiently expensive, increase anesthesia cost, the most most of laboratorys are difficult to push away
Wide application.
Animal Anesthesia development trend is balanced anesthesia at present, namely multi-medicament use in conjunction, not only
Can reasonable control depth of anesthesia, reach operation needs, it is also possible to reduce single medicine use
Amount, reduces drug accumulation, alleviates the excessive side effect caused of drug level.Therefore, for various notes
Penetrating the action character of anaesthetic, preparation animal specific compound preparation is that the main development of balanced anesthesia becomes
Gesture.Further, since animal has wild nature and non-domestication, operate and loaded down with trivial details cause people and animals to damage unavoidably.
Therefore, the compound anesthetic that energy one pin proves effective it is badly in need of.And most with prospects be balanced anesthesia compound recipe
Narcotic research, is growing point new in animal medicine anesthesia field, current existing procucts for dog,
In the anaesthesia process of the animals such as deer.
Existing rat anesthesia and Baoding remain in the use of above-mentioned single medicine, the discussion of dosage and
Several drugs be compared to each other the stage, need a kind of new compound recipe preparation for callouse badly, solve existing rat
Deficiency that anesthetis exists at the aspect such as calmness, analgesia, induction time, anesthesia maintenance time and big
The outstanding problems such as rapid heart rate that Mus irritability is excessive to be caused, hyperpiesia.
Summary of the invention
An object of the present invention is to provide a kind of rat compound anesthetic；
The two of the purpose of the present invention are to provide the preparation method of a kind of rat compound anesthetic.
The above-mentioned purpose of the present invention is achieved through the following technical solutions:
Present invention firstly provides a kind of rat compound anesthetic, including: dexmedetomidine, miaow
Reach azoles logical sequence, oxycodone, buffer and water for injection；
Wherein, described buffer is preferably the phosphate buffer of pH7.0.
First the multiple component with anaesthetic effect being applied to animal is carried out random combine by the present invention,
By the contrast test to rat anesthesia, comprehensive analyze the Anesthesia induction of various combinations, anesthetic stage,
The physical signs such as waking up period, calmness, analgesia, flesh pine effect and breathing, pulse, body temperature；Pass through
Substantial amounts of screening experiment is it was found that by answering that dexmedetomidine, midazolam and oxycodone form
The anaesthetic effect of square preparation is substantially better than the anaesthetic effect of other various combinations.Therefore, present invention determine that
Main using dexmedetomidine, midazolam and oxycodone as the compound recipe preparation for callouse of rat
Component.
Dexmedetomidine is a kind of novel high selectivity α23 adrenergic receptor agonists, it swashs
Live nucleus ceruleus α2Receptor, and nucleus ceruleus is α in brain2The region that A receptor is the most intensive, is negative in brain
Duty reconciles the key position of awakening and sleep, thus produces nature non-dynamic eye sleep (NREM), sends out
Wave calmness, angst resistance effect.And dexmedetomidine is slight to respiration inhibition, its metabolism is by renal function
Affecting little, metabolite does not has activity.Dexmedetomidine has the analgesic activity of moderate, simultaneously its tool
Some calmness, minimizing stress and the synergism etc. of opioid drug, it is possible to reduce perioperative
The consumption of opioid drug.Quickly give 1 μ g/kg dexmedetomidine of loading dose, directly swash
Live the α in vascular smooth muscle2A receptor, produces vasoconstriction, can cause of short duration hypertension, reflection
Property ground reduce heart rate.Dexmedetomidine can deepen rapidly general anesthesia, and anesthesia speed is accelerated, and has it
Specific antagonist medicine, it is simple to the regulation general anesthesia degree of depth, controllability is better than most general anesthetics.
After midazolam induction of anesthesia, heart rate variability (HRV) each composition all reduces, nor-kidney in blood plasma
The concentration of upper parathyrine (NA) substantially reduces, and has relatively for its irritability and dexmedetomidine
Good synergism.Intravenous midazolam may result in blood pressure drops, directly suppresses cardiac muscle, expansion periphery
Blood vessel and affect autonomic nerve.Dexmedetomidine is the most more apparent on the impact of heart rate, blood pressure.The two
In the impact slowing down anesthetic stage laboratory animal heart rate, there is synergism.Oxycodone is from the alkaloid base of a fruit
The semi-synthetic opioid drug extracted in Ba Yin (Thebaine), as powerful analgesics clinically
The application history of existing more than 80 year.Owing to oxycodone bioavailability is high, route of administration is many, thus
Clinical medicine is widely used, in terms of Animal Anesthesia, is rarely employed this medicine.
Determining using dexmedetomidine, midazolam and oxycodone as rat compound recipe anesthesia system
On the basis of the key component of agent, the present invention use to dexmedetomidine, midazolam and oxycodone
The buffer salt system of amount and compound anesthetic is further optimized and is screened.
The consumption of dexmedetomidine, midazolam and oxycodone is screened and obtains anaesthetic effect by the present invention
Preferable 3 consumptions combination, as prescription 1,2 and 3.
Wherein, in prescription 1, each component and content are: in compound anesthetic described in every 100mL, right
The content of rotation dexmedetomidine is 15mg, and the content of midazolam is 135mg, and the content of oxycodone is
6.5mg。
In prescription 2, each component and content are: in compound anesthetic described in every 100mL, dextrorotation U.S. holds in the palm
The content of miaow pyridine is 15mg, and the content of midazolam is 145mg, and the content of oxycodone is 7.5mg.
In prescription 3, each component and content are: in compound anesthetic described in every 100mL, dextrorotation U.S. holds in the palm
The content of miaow pyridine is 15mg, and the content of midazolam is 135mg, and the content of oxycodone is 7mg.
Experimental result finds, although prescription 3 does not has substantially with prescription 1 and 2 in calm and flesh loose measure face
Difference, but prescription 3 is significantly better than prescription 1 and 2 in terms of analgesic effect.Thereby determine that the best of it
Side is prescription 3, and in compound anesthetic described in i.e. every 100mL, the content of dexmedetomidine is
15mg, the content of midazolam is 135mg, and the content of oxycodone is 7mg.
Therefore, in order to reach more preferably anaesthetic effect, in compound anesthetic described in every 100mL, respectively
The content of component is preferably: in every 100mL compound anesthetic, dexmedetomidine 15mg, miaow reaches
Azoles logical sequence 135mg, oxycodone 7mg；Or in every 100mL compound anesthetic, the content of each component
For: dexmedetomidine 15mg, midazolam 135mg, oxycodone 6.5mg；Or every 100mL
In compound anesthetic, the content of each component is: dexmedetomidine 15mg, midazolam 145mg,
Oxycodone 7.5mg.
When preparing compound anesthetic, use different buffer salt system, its anaesthetic effect (include calmness,
Flesh pine, analgesia etc.) there is bigger difference, optimization of the present invention, screen different buffer, finally
Find, use the anesthetis of phosphate buffered saline of pH7.0 in anesthesia such as calm, flesh pine or analgesias
Other buffer is substantially better than in effect.Therefore, the present invention uses the phosphate buffer of pH7.0
Preparation compound anesthetic；Wherein, the phosphate buffer of described pH7.0 includes: potassium dihydrogen phosphate and
Sodium hydroxide；Preferably, in every 100mL compound anesthetic: the content of potassium dihydrogen phosphate is 680mg,
The content of sodium hydroxide is 116.4mg.
The present invention is also to whether adding atropine and the joining day is tested, it was found that and compound recipe
The analgesic effect in anaesthesia process can be strengthened after narcotic key component injecting atropine simultaneously, it is also possible to
Effectively improve compound recipe preparation for callouse to rat heart rate and the harmful effect of blood pressure.Finally determine atropine and
The key component of compound anesthetic is simultaneously introduced, and dosage is 0.14mg/kg, i.e. every 100mL compound recipe anesthesia
In agent, atropinic content is 14mg.
Atropine belongs to postganglionic anticholinergic drug, blocks muscarinic receptor, there are some researches show that atropine can
So that the effect that heart rate is increased by nicotine decreases, its reason is probably That atropine blocks nicotine thorn
Swash cardiac sympathetic nerve tip release acetylcholine.The present invention select atropine as adjuvant, be because of
Atomic little on arterial pressure impact for this medicine, the impact on heart rate also relaxes far beyond epinephrine.It is the brightest
Aobvious effect is the impact on heart rate, and this effect shows as " biphasic reaction " sometimes, i.e. after administration
Just may occur in which of short duration decreased heart rate, then heart rate speedup.Its accelerating function and original heart rate level
In negative correlation, utilize this effect to relax dexmedetomidine and heart rate is reduced amplitude, play protection dynamic
The effect of thing body.
Wherein, heretofore described murine is preferably rat.
The invention provides a kind of method preparing described murine compound anesthetic, the method bag
Include: dexmedetomidine, midazolam, oxycodone, buffer are dissolved in water for injection, dilution,
Filter, sterilizing, to obtain final product.
Present invention also offers a kind of method preparing described murine compound anesthetic, the method
Including: dexmedetomidine, midazolam, oxycodone, atropine, buffer are dissolved in note successively
Penetrate with in water, dilution, filter, embedding in ampoule bottle, sterilizing, to obtain final product.
The animal of present invention compound anesthetic can be applied to braking and the fiber crops of animal especially rat
Liquor-saturated.
The animal of present invention compound anesthetic has the advantage that compared with existing Animal Anesthesia agent
1, the induction of anesthesia time is 6.35 ± 2.28min, and the anesthesia maintenance time is 130.23 ± 57.22min,
Recovery time is 33.28 ± 17.05min.Show that the induction of anesthesia of this compound anesthetic is steady rapidly, anesthesia
Hold time length, revive steadily, performance without exception (excited, sleep again).
2, the impact on major physiological parameter: inject this compound anesthetic has fixing to normal body temperature
Ringing, after showing as injection, body temperature begins to decline and persistently maintains low temperature state.Heart rate is after injection
During 7.5min compared with basic value, significantly reduce, use atropine can effectively alleviate the excessive of heart rate
Reduce and arrhythmia etc..In slightly decreasing trend after respiratory frequency injection, one factor analysis of variance is poor
Different not notable, substantially recover normal level after lasting till anesthesia 120min.Blood oxygen saturation shows as
Initial stage decline, after injection, 20min is down to minimum, after gradually recover and close to normal level, whole
In anaesthesia process, blood oxygen saturation is all more than 93%.Blood pressure starts to raise, to 10min after injection
Maintain hypertensive state later, be gradually lowered after 40min, blood pressure and 0min diversity after 80min
The most not notable (p > 0.05).Inject this compound anesthetic resistance at its physiology on Study on Physiologic impact
In the range of being subject to.
3, the impact on hepatic and renal function: ALT, AST, ALP, TBIL, BUN, CREA,
, in anaesthesia process, there is not significant change in the Main Biochemical of the reflection hepatic and renal function such as CHOL
(p>0.05)。
4, simple to operate, lumbar injection, anaesthetize for rapid induction；The anesthesia maintenance time is longer,
Analgesia, sedation effect well, can be used for complexity or the operation of time length, and recovery time is very fast.
Accompanying drawing explanation
Fig. 1 is that three kinds of lower heart rate averages of process change over trend；It is (secondary that the longitudinal axis represents heart rate
/ min), horizontal axis plots time point (min).
Fig. 2 be under three kinds of different disposal mean arterial pressure (MAP changes over trend；Longitudinal axis generation
Table blood pressure, horizontal axis plots time point (min).
Fig. 3 is to shrink pressing mean under atropine various dose to change over trend；The longitudinal axis represents
Blood pressure, horizontal axis plots time point (min).
Detailed description of the invention
Further describing the present invention, advantages of the present invention and feature below in conjunction with specific embodiment will be with
Description and apparent.It should be understood that described embodiment is only exemplary, not to the present invention's
Scope constitutes any restriction.It will be understood by those skilled in the art that in the essence without departing from the present invention
The details of technical solution of the present invention and form can be modified or replace under god and scope, but these
Amendment or replacement each fall within protection scope of the present invention.
The preparation of embodiment 1 compound anesthetic
In compound anesthetic, each component and content are: in every 100mL compound anesthetic, each component contains
Amount is dexmedetomidine 15mg, midazolam 135mg, oxycodone 7mg, potassium dihydrogen phosphate
680mg, sodium hydroxide 116.4mg, surplus is water for injection.
The preparation method of compound anesthetic, comprises the following steps: by dexmedetomidine, midazolam,
Oxycodone, potassium dihydrogen phosphate, sodium hydroxide are dissolved in water for injection successively, add water for injection dilute
Releasing, filter, embedding is in ampoule bottle, through autoclaving, to obtain final product.
The preparation of embodiment 2 compound anesthetic
In compound anesthetic, each component and content are: in every 100mL compound anesthetic, each component contains
Amount is dexmedetomidine 15mg, midazolam 135mg, oxycodone 7mg, atropine 14mg,
Potassium dihydrogen phosphate 680mg, sodium hydroxide 116.4mg, surplus is water for injection.
The preparation method of compound anesthetic, comprises the following steps: by dexmedetomidine, midazolam,
Oxycodone, atropine, potassium dihydrogen phosphate, sodium hydroxide are dissolved in water for injection successively, add note
Penetrating dilute with water, filter, embedding is in ampoule bottle, through autoclaving, to obtain final product.
The preparation of embodiment 3 compound anesthetic
In compound anesthetic, each component and content are: in every 100mL compound anesthetic, each component contains
Amount is dexmedetomidine 15mg, midazolam 135mg, oxycodone 6.5mg, and surplus is injection
Use water.
The preparation method of compound anesthetic, comprises the following steps: by dexmedetomidine, midazolam,
Oxycodone is dissolved in water for injection successively, adds water for injection dilution, filters, and embedding is in ampoule bottle
In, through autoclaving, to obtain final product.
Laboratory animal
SD rat 180, at the 2-2.5 monthly age, male and female half and half, body weight 258 ± 23g, by Heilongjiang Province
University of TCM's Experimental Animal Center provides, and raises 4 weeks and test under identical conditions, whole examination
During testing, feeding and management condition keeps consistent.
SJL strain white mice 60,1 monthly age, female, body weight 22 ± 3g is big by Harbin medical courses in general
Experimental Animal Center provides, and tests after raising 2 weeks under identical conditions.
White rabbit 4, at 1 monthly age, male and female do not limit, and body weight is respectively 1.8kg and 2.1kg, by Heilungkiang
University of TCM's Experimental Animal Center provides, and tests after raising 2 weeks under identical conditions.
The component of experimental example 1 compound anesthetic and consumption screening experiment
One, the medicament categories initial screening experiments of compound anesthetic
(1) experimental technique
1. sedation effect monitoring and evaluation criterion
After rat anesthesia, after observing medication respectively, calmness when 5min, 10min, 20min and 30min is imitated
Really.Observe nictation, ear is dynamic, body is dynamic, head and neck is dynamic, tail moves.
To sonic stimulation significant reaction, before being administered, note "+"；To sonic stimulation bradykinesia, remember " ± "；
Reactionless to sonic stimulation, remember "-".
Methods of marking: "+" meter 0 point, " ± " meter 1 point, "-" meter 2 points.
2. analgesic effect monitoring and evaluation criterion
Mainly it is evaluated by acupuncture muffle portion, trunk, extremity top, claw, afterbody.
Acupuncture each portion pain reaction is obvious, note "+"；Pain reaction is blunt, note " ± "；Pain reaction disappears,
Note "-".
Methods of marking: "+" meter 0 point, " ± " meter 1 point, "-" meter 2 points.
3. flesh pine effect monitoring and evaluation criterion
Viewing head cervical region, tail, extremity, masseter and abdominal wall muscle relax degree.
As there is later and turn round reaction when mentioning with tweezers folder tail, extremity appearance is substantially slided, and stimulates four
Limb still can be creeped, note "+"；Folder tail occurs that rotary head, simultaneously extremity slightly slide when mentioning, note " ± "；Nothing
Turn one's head and turn round reaction, remembering "-".
Folder ear when mentioning as tail be flexible to horizontal level or upwarp time, remember "+"；As tail occurs that bending is anti-
Should, note " ± "；Tail naturally droops, and remembers "-".
Clamp ear with tweezers to mention, as struggling occur in extremity, note "+"；Extremity slightly slide, note " ± "；
Extremity naturally droop, and remember "-".
If postabdomen is without significant change when folder ear is mentioned, remember "+"；Postabdomen slightly expands lax, note " ± "；
Postabdomen substantially expands lax, remembers "-".
Masseter is lax gently to be pulled on, lower jaw, oral cavity can smooth opening, note "+"；Oral cavity can be opened, but very
Soon can recuperator, note " ± "；Recuperator immediately after can not opening or open, remembers "-".
Methods of marking: "+" meter 0 point, " ± " meter 1 point, "-" meter 2 points.
(2) experimental result
When experimental result in table 1 is 20min, (now anaesthetic effect is relatively stable) often group owns
The score summation (individual event highest score is 20 × 10 points=200 points) of experimental animal.
20min rat anesthesia effect observation (n=20) after table 1 injection
First preferably gone out dexmedetomidine by table 1 and enter next step test as principal agent.
20min rat anesthesia effect observation (n=20) after table 2 injection
Dexmedetomidine+fentanyl and the effect of dexmedetomidine+oxycodone is can be seen that by table 2
Fruit preferably, but comprehensively analyze the cost of medicine, additive, toxic and side effects and drug control degree etc. because of
Element, then select dexmedetomidine+oxycodone and enter next step experiment.
20min rat anesthesia effect observation (n=20) after table 3 injection
Can be seen that dexmedetomidine+fentanyl+midazolam effect is best, particularly by table 3
Have better effect at ease pain, therefore select dexmedetomidine+oxycodone+midazolam as compound recipe fiber crops
The main component of liquor-saturated dose.
By the contrast test to rat anesthesia, comprehensive the analysis Anesthesia induction of various combinations, fiber crops
Liquor-saturated phase, waking up period, the physical signs such as calm, analgesia, flesh pine effect and breathing, pulse, body temperature,
Find that the compound preparation being made up of dexmedetomidine, midazolam and oxycodone is substantially better than other each
Plant combination, therefore tentative using dexmedetomidine, midazolam and oxycodone as compound recipe preparation for callouse
Main component.
Two, the usage ratio explorative experiment of each component of compound anesthetic
(1) drug dose initial screening experiments method
(1) different pharmaceutical ED50Mensuration
Experimental design arranges drug dose by geometric progression, and making adjacent two dosage logarithmic differences is 0.05.
Test from estimating that most probable is close to ED50Dosage start, repeatedly explored by preliminary experiment, find
May be close to ED50Dosage, then each prescription medicinal liquid is diluted to 15mL, it is ensured that middle be administered
Dosage is in the range of 0.010～0.015mL/g, it is ensured that ip dosage is within claimed range.Real
Carry out according to one ground of sequential method principle during testing, when positive reaction occurs in a rat
(righting reflex loss) then next rat is just with the dosage of low one-level, on the contrary, when feminine gender occur
Higher leveled dosage is then used during reaction.Statistical experiment animal is started when first set reaction sign modification
Number (partial results is shown in Table 4).
(2) different prescription LD50Mensuration
Composition formula LD50Assay method and ED50Mensuration roughly the same, difference be measure
LD50Observed positive indication is dead.
Table 4 different pharmaceutical ED50And LD50Mensuration
(3) anaesthetic effect methods of marking
Specific experiment method sees in above-mentioned " the medicament categories initial screening experiments of compound anesthetic "
Experimental technique.
(2) experimental result
Dexmedetomidine, midazolam and three kinds of medicines of oxycodone are determined by the assay method of table 4
ED50For 0.18mg/kg, 1.00mg/kg, 0.07mg/kg.In this, as drug regimen level 1,
Increase by the using dosage of 20% and 40% afterwards, and press the Three factors-levels various combinations of mensuration respectively
Anaesthetic effect, concrete outcome is shown in Table 5 and table 6.
Table 5 factor level table
Table 6 white mice anaesthetic effect scoring (after three kinds of medicine composite injections, 20min marks, n=20)
Note: data compare the different capitalization of shoulder mark and represent significant difference (P ＜ 0.05)；Shoulder is marked identical capitalization and is represented that difference is not
Significantly (P ＞ 0.05).
With the various combination being made up of dexmedetomidine, midazolam and oxycodone, rat is carried out abdomen
Chamber is injected, and observes the situations such as biology reflection, calmness, analgesia, flesh pine, and the anesthesia to various combination is made
With comprehensively analyzing and evaluating, Preliminary screening goes out with dexmedetomidine, midazolam, oxycodone
As the component of compound preparation, and draft the preliminary dosage of three be 0.20mg/kg, 1.4mg/kg,
0.07mg/kg。
Three, orthogonal test
According to Three factors-levels Orthogonal Experiment and Design principle and combine the sight of anaesthetic effect in trial test
Examine and be designed.3 factors in compound recipe preparation for callouse are dexmedetomidine, midazolam, hydroxyl
Examining ketone, 3 levels are high, medium and low (table 7).According to orthogonal table L9 (33) by white mice with
Machine is divided into 9 test group, the samples selection principle in designing according to statistics standard and pharmacology, each
Test group selects white mice 30, lumbar injection 0.01mL/g, analyzes each medicine group according to orthogonal table
Divide the impact on anaesthetic effect.
Table 7 factor level table
After white mice anesthesia, righting reflex loss reaches at more than 1min person is judged to the positive in 30min.
As shown in Table 8: dexmedetomidine effect when low dosage is better than middle and high dosage；Miaow reaches azoles
Logical sequence effect when low dosage is better than dosage high, middle；Oxycodone effect when middle dosage is better than high and low dose
Amount.Comparative analysis experimental result, determines that best of breed is: dexmedetomidine 0.15mg/kg, miaow
Reach azoles logical sequence 1.35mg/kg, oxycodone 0.07mg/kg.
Table 8 white mice Orthogonal experiment results (n=30)
Note: * represents maximum in each column three level list drug effect fruit, and E1, E2, E3 are the drug effect sum of the basic, normal, high level of this medicine.
Select the best experiment group 1 of the righting reflex effect in table 8, group 3 respectively as group
Side 1 and prescription 2, by best of breed (dexmedetomidine 0.15mg/kg, midazolam 1.35
Mg/kg, oxycodone 0.07mg/kg) enter next step orthogonal decisive confirmatory experiment as prescription 3.
Four, orthogonal decisive confirmatory experiment
1 experimental technique
Laboratory animal SD rat is randomly divided into 3 groups, i.e. group 1, group 2 and group 3；Often group 20；
Group 1, group 2 and group 3 are respectively with the compound anesthetic 1 prepared by prescription 1, answering by prescription 2 preparation
Side's anesthetis 2 and the compound anesthetic 3 prepared by prescription 3 carry out anaesthetizing (consumption of administration, approach and time
Etc. identical), observe the calmness after each group of rat anesthesia is administered, analgesia, pine flesh effect respectively.
Wherein, consisting of of the compound anesthetic 1 that group 1 uses: in compound anesthetic described in every 100mL,
The content of dexmedetomidine is 15mg, and the content of midazolam is 135mg, and the content of oxycodone is
6.5mg；
Consisting of of the compound anesthetic 2 that group 2 uses: in compound anesthetic described in every 100mL, dextrorotation
The content of dexmedetomidine is 15mg, and the content of midazolam is 145mg, and the content of oxycodone is
7.5mg；
Consisting of of the compound anesthetic 3 that group 3 uses: in compound anesthetic described in every 100mL, dextrorotation
The content of dexmedetomidine is 15mg, and the content of midazolam is 135mg, and the content of oxycodone is 7mg.
2 experimental results
Experimental result is shown in Table 9；Can be seen that from the experimental data of table 9, although by the compound recipe fiber crops of prescription 3 preparation
Especially significant difference is not had with by the compound anesthetic of prescription 1 and 2 preparation for liquor-saturated dose 3 in calm and flesh loose measure face
, but at ease pain, not to be significantly better than by the compound anesthetic 3 of prescription 3 preparation and prepare by prescription 1 and 2
Compound anesthetic 1 and 2, prescription 3 has exceeded 16.8% and 16.3% respectively than prescription 1 and prescription 2.Thus
Determine that best prescription is prescription 3.The each component of prescription 3 and content is: in compound anesthetic described in every 100mL,
The content of dexmedetomidine is 15mg, and the content of midazolam is 135mg, and the content of oxycodone is
7mg。
Table 9 rat anesthesia effect observation (n=20)
The buffer salt system screening experiment of experimental example 2 compound anesthetic
1, experimental technique
1.1 experiment packets, preparation containing buffer salt compound anesthetic
DZ group: sterile saline 1000ml.
JS group (citrate buffer pH6.2): take 2.1% aqueous solution of citric acid, use 50% sodium hydroxide
Solution regulation pH value, to 6.2, to obtain final product.
Citric acid--disodium hydrogen phosphate buffer (pH4.0).
Solution A: take citric acid 21g or anhydrous citric acid 19.2g and add water and make to be dissolved into 1000ml, put in refrigerator
Preserve.
Second liquid: take disodium hydrogen phosphate 71.63g, adds water and makes to be dissolved into 1000ml.Take above-mentioned solution A 61.45ml
Mix with second liquid 38.55ml and shake up, to obtain final product.
BL group (barbital-sodium chloride buffer pH7.8): take barbital sodium 10.1g, add sodium chloride 7.4g
And water dissolves in right amount, separately take gelatin 1g and add water in right amount, be incorporated to after heating for dissolving in above-mentioned solution.Then use
0.2mol/L hydrochloric acid solution regulation pH value, to 7.8, adds water and is diluted to 1000ml, to obtain final product.
LS group (phosphate buffer, pH7.0): take potassium dihydrogen phosphate 6.8g and add 0.1mol/L sodium hydroxide
Solution 291ml, is diluted with water to 1000ml, to obtain final product.
Compound anesthetic is prepared by water for injection and above-mentioned buffer salt system.
The each component of compound anesthetic and content is: in every 100mL compound anesthetic, the content of each component is
Dexmedetomidine 15mg, midazolam 135mg, oxycodone 7mg.
The preparation method of compound anesthetic, comprises the following steps: by dexmedetomidine, midazolam,
Oxycodone is dissolved in buffer salt system successively, mixing of fully vibrating, and adds water for injection dilution, mistake
Filter, embedding in ampoule bottle, autoclaving, both.Keep in Dark Place in 4 DEG C.
40 white mice are divided into 4 groups at random, i.e. DZ, LS, JS and BL group, often group lumbar injection phase
The compound anesthetic 0.01mL/g of buffer salt system preparation should be organized, enter anaesthetic effect monitoring experiment afterwards.
The anaesthetic effect monitoring of 1.2 white mice
1.2.1 sedation effect monitoring
After white mice anesthesia, observe calmness when 5min, 15min, 30min and 60min after medication respectively
Effect.Observe nictation, ear is dynamic, body dynamic, head and neck is dynamic, extremity and tail move.
1.2.2 analgesic effect monitoring
After white mice anesthesia, observe analgesic effect when 5min, 15min, 30min and 60min after medication.
Mainly it is evaluated by acupuncture muffle portion, trunk, extremity top, claw, afterbody.
1.2.3 flesh pine effect monitoring
After white mice anesthesia, observe flesh pine effect when 5min, 15min, 30min and 60min after medication.
Viewing head cervical region, tail, extremity, masseter and abdominal wall muscle relax degree.
Sedation effect, analgesic effect and flesh pine effect assessment standard are real with decisive checking orthogonal in experimental example 1
Test.
After white mice anesthesia, righting reflex loss reaches at more than 1min person is judged to the positive in 30min, according to
Upper standard is evaluated, and reference is calm, ease pain and the harmony of flesh pine effect carries out the comprehensive of anaesthetic effect
Evaluation.
1.3 irritation test
1.3.1 intramuscular injection irritation test
Take healthy rabbit 4, at the quadriceps femoris of side, inject each group of water for injection or buffer salt body
Being the compound anesthetic lmL as solvent preparation, it is raw that opposite side corresponding site injects equal-volume sterilizing with method
Reason saline is as comparison.After 48h, sacrificed by exsanguination white rabbit, take out quadriceps femoris after dissection, longitudinally slit,
Observe the muscle package irritant reaction of injection site, and be converted into by table 4 and react score value accordingly.
Table 10 intramuscular injection irritation test criterion
1.3.2 eye drip irritation test
Taking healthy rabbit 4, Baoding, check two, person without exception can be used for testing.Open gently,
Palpebra inferior, is respectively dropped into each to each group of medicinal liquid and normal saline 2 in images of left and right eyes conjunctival sac, two
Instilling dose should be equal, eyelid about 2～3s of the gentliest sleeping, in case medicinal liquid flows out affects result of the test.Observe
After both sides ocular administration and normal saline 10,20,30,60,90 and 120min time eye conjunctiva change.
2, experimental result
The anaesthetic effect monitoring of 2.1 white mice
The anaesthetic effect of white mice the results are shown in Table 11, table 12, table 13 and table 14, it can be seen that fiber crops
Liquor-saturated early stage (5min after injection), anesthesia mid-term (15min and 30min after injection), LS group white mice
Anaesthetic effect is best, and calmness, flesh pine, analgesic effect are substantially better than other three groups (P ＜ 0.05).Fiber crops
In the liquor-saturated later stage (60min after injection), the calmness of LS group, analgesic effect are substantially better than other three groups (P ＜ 0.05).
The observation (n=10) of anaesthetic effect after table 11 white mice injection 5min
Note: compare between group, data shoulder marking-up mother's difference represents significant difference (P ＜ 0.05).
The observation (n=10) of anaesthetic effect after table 12 white mice injection 15min
Note: statistical analysis labelling is with table 9.
The observation (n=10) of anaesthetic effect after table 13 white mice injection 30min
Note: statistical analysis labelling is with table 9.
The observation (n=10) of anaesthetic effect after table 14 white mice injection 60min
Note: statistical analysis labelling is with table 9.
2.2 irritation test
Intramuscular injection irritation test result shows, four groups of injection points and offside physiological saline point zero difference,
Injection point meat color is normal, and the reaction score value of 4 rabbit is all cited as 0 point.Different buffer salt is described
The compound anesthetic of system meets required standard, is available for intramuscular injection.
Eye drip irritation test result shows, after four groups of eye dripping, during only JS and BL group 10min, and eye
Conjunctiva presents slight flushing, tendency of shedding tears.During 25min, bulbar conjunctiva starts flushing, hyperemia occur,
And show slight photophobia, phenomenon of shedding tears.During to 40min, flushing disappears, and eye conjunctiva color and luster is substantially
Recover state to medication, have no that other is abnormal.LS group and comparison DZ group then within the whole observation period,
If unsighted conjunctiva has ANOMALOUS VARIATIONS.
Based on the above results, determine that optimized buffer salt system is LS group (phosphate buffer, pH7.0),
Compound anesthetic is prepared as buffer system.
The experimental example 3 compound anesthetic of the present invention Anesthetic Effect to rat
1, for examination anesthetis
Compound anesthetic includes: dexmedetomidine, midazolam, oxycodone, potassium dihydrogen phosphate,
Sodium hydroxide and water for injection；The content of each component is: each component in every 100mL compound anesthetic
Content be dexmedetomidine 15mg, midazolam 135mg, oxycodone 7mg, di(2-ethylhexyl)phosphate
Hydrogen potassium 680mg, sodium hydroxide 116.4mg, surplus is water for injection.
The preparation method of compound anesthetic, comprises the following steps: by dexmedetomidine, midazolam,
Oxycodone, potassium dihydrogen phosphate, sodium hydroxide are dissolved in water for injection successively, add water for injection dilute
Release, filter, embedding in ampoule bottle, autoclaving, both.
2, experimental technique and result
2.1 anesthesia
Weigh rat body weight.Put into blood pressure holder until its adapt to after, measure its basic physiology by tail sleeve method
Index such as heart rate (HR), mean arterial pressure (MBP) etc..Rat is put into holder after its peace and quiet
Measure its physiological indexes and include respiratory frequency (RR), arterial oxygen saturation (SpO2), body temperature (T)
Deng.Press 1mL/kg dosage lumbar injection compound anesthetic afterwards, Monitored anesthesia period, calmness, analgesia
And flesh pine effect.Take heating to measure rat non-invasive blood pressure, connect AD Instruments and lead life more
Reason monitor and MP150 type 16 passage physiological signal instrument carry out heart rate, ecg wave form, blood oxygen saturation,
The index such as body temperature, respiratory frequency is monitored continuously, notes heating in good time and insulation in experimentation.
Respectively at different time points by cardiac acquisition venous blood 0.5mL, after placing 1h, 1500rpm
Centrifugal 10min, separates the serum without chyle and haemolysis and manages to EP.AU5800 series full-automatic biochemical divides
Analyzer measures its glutamate pyruvate transaminase ALT (U/L), glutamic oxaloacetic transaminase, GOT AST (U/L), alkali phosphatase
ALP (U/L), total bilirubin TBIL (mg/L), blood urea nitrogen BUN (mg/L), creatinine CREA
(mg/L), the Main Biochemical of the reaction such as T-CHOL CHOL (mg/L) hepatic and renal function.
2.2 monitorings anaesthetizing period
Anesthesia induction is 6.35 ± 2.28min, and the anesthesia maintenance time is 130.23 ± 57.22min, revives
Time is 33.28 ± 17.05min, revives steadily, performance without exception (excited, sleep again).
2.3 anaesthetic effects (analgesia, calm, flesh pine) monitoring
Empirically in example 1, the evaluation criterion in orthogonal decisive confirmatory experiment is marked, and anaesthetic effect obtains
It is divided into every score sum (table 15).
Table 15 rat anesthesia effect Score Lists (n=10)
Note: shoulder mark capitalization difference represents significant difference, P ＜ 0.05.
2.4 physiological functional moni-toring
Injection compound anesthetic has certain impact to rat normal body temperature, under after showing as injection, body temperature starts
Fall also persistently maintains low temperature state.Heart rate, significantly reduces when 7.5min after injection compared with basic value.
In slightly decreasing trend after respiratory frequency injection, one factor analysis of variance difference is not notable, lasts till anesthesia
Substantially normal level is recovered after 120min.Blood oxygen saturation shows as initial stage decline, 20min after injection
Be down to minimum, after gradually recover and close to normal level, in whole anaesthesia process, blood oxygen saturation is all 93%
Above.Blood pressure start after injection raise, after 10min, maintain hypertensive state, after 40min by
Gradually reducing, after anesthesia, 15min-80min blood pressure and 0min significant difference (p ＜ 0.05), poor after 80min
The opposite sex is not the most notable (p > 0.05), is shown in Table 16.
Every physiological parameter change (n=10) of rat after table 16 anesthesia
Note: compare with 0min, * represents significant difference, P ＜ 0.05.
2.5 hepatic and renal function monitorings
Compound anesthetic is on rats'liver renal function without significantly impact, and concrete outcome is shown in Table 17.
Rats'liver renal function index change (n=10) after table 17 anesthesia
Note: compare with 0h, * represents significant difference, P ＜ 0.05.
Result above shows, the induction of anesthesia of this compound anesthetic is steady rapidly, and the anesthesia maintenance time is long,
Revive steadily, performance without exception (excited, sleep again).Anaesthetic effect is good, analgesia, sedation effect
Well, main physical signs is affected in the range of its physiological tolerance, to rats'liver renal function without substantially
Impact.
Experimental example 4 adds atropine experiment
On the basis of the key component and ratio-dependent of compound recipe preparation for callouse, to whether add atropine and
The atropine joining day tests.
Compound anesthetic includes: dexmedetomidine, midazolam, oxycodone, potassium dihydrogen phosphate,
Sodium hydroxide and water for injection；The content of each component is: each component in every 100mL compound anesthetic
Content be dexmedetomidine 15mg, midazolam 135mg, oxycodone 7mg, di(2-ethylhexyl)phosphate
Hydrogen potassium 680mg, sodium hydroxide 116.4mg, surplus is water for injection.
Take SD rat 24 and be divided into A, B, C tri-groups at random, press 1mL/kg dosage abdominal cavity afterwards
Injection compound anesthetic, wherein A group is without atropine；B group is 15min injecting atropine in advance
(0.14mg/kg)；C group is and compound anesthetic injecting atropine (0.14mg/kg) simultaneously.
Experimental result finds, can strengthen anesthesia after the key component injecting atropine simultaneously of compound anesthetic
During analgesic effect (table 18), it is also possible to effectively improve compound recipe preparation for callouse to rat heart rate (table
19, table 20, Fig. 1) and the harmful effect of blood pressure (table 21, table 22, Fig. 2), especially relax multiple
Heart rate is reduced amplitude by side's anesthetis.Simultaneously after injecting atropine, induction of anesthesia time and recovery time are relatively
Group without atropine, in advance 15min injecting atropine group no significant difference (table 23).Finally determine atropic
The key component of product and compound anesthetic is simultaneously introduced, and dosage is 0.14mg/kg, i.e. every 100mL compound recipe
In anesthetis, atropinic content is 14mg.
From heart rate trend over time, add simultaneously atropinic rate stability be better than other two
Group, the most further in the case of atropine is administered simultaneously, adds dosage to atropine and inquires into.
When anaesthetizing with reference to dog, atropinic consumption and preliminary result, try after determining using dosage variable gradient
Test.Result display atropine is after medication 15min, and blood pressure raises notable (table 24).
Table 18 rat anesthesia effect observation (n=8)
The monitoring result of table 19 basal heart rate (N=8)
Changes in heart rate situation under 20 3 kinds of different disposal of table (N=8)
Note: * represents variant with basal heart rate, and under in table, same letter represents same time conditions, the two difference is not notable, and different letter representation significant differences are aobvious
Work level is 0.05.
The monitoring result of table 21 basic blood pressure (N=8)
Mean arterial pressure (MAP) monitoring result under 22 3 kinds of different disposal of table (N=8)
Note: * represents variant with basic blood pressure, and under in table, same letter represents same time conditions, the two difference is not notable, and different letter representation significant differences are aobvious
Work level is 0.05.
Induction time and recovery time (n=8) under 23 3 kinds of different disposal of table
Shrink pressure under table 24 atropine various dose and diastolic pressure average changes over table (n=8)
Claims (7)
1. a rat compound anesthetic, it is characterised in that consist of the following composition: dextrorotation is beautiful
Torr miaow pyridine, midazolam, oxycodone, buffer and water for injection；
In every 100mL compound anesthetic, the content of each component is: dexmedetomidine 15mg, miaow
Reach azoles logical sequence 135mg, oxycodone 7mg；Or in every 100mL compound anesthetic, containing of each component
Amount is: dexmedetomidine 15mg, midazolam 135mg, oxycodone 6.5mg；Or every 100mL
In compound anesthetic, the content of each component is: dexmedetomidine 15mg, midazolam 145mg,
Oxycodone 7.5mg.
2. a rat compound anesthetic, it is characterised in that consist of the following composition: dextrorotation is beautiful
Torr miaow pyridine, midazolam, oxycodone, atropine, buffer and water for injection；
Wherein, in every 100mL compound anesthetic, the content of each component is: dexmedetomidine 15mg,
Midazolam 135mg, oxycodone 7mg, atropine 14mg.
3. according to the rat compound anesthetic described in claim 1 or 2, it is characterised in that: institute
The buffer stated is the phosphate buffer of pH 7.0.
4. according to the rat compound anesthetic described in claim 3, it is characterised in that: phosphate
Buffer includes potassium dihydrogen phosphate and sodium hydroxide；Wherein, in every 100mL compound anesthetic, phosphorus
The content of acid dihydride potassium is 680mg, and the content of sodium hydroxide is 116.4mg.
5. prepare the method for rat compound anesthetic described in claim 1 for one kind, it is characterised in that:
Dexmedetomidine, midazolam, oxycodone, buffer are dissolved in water for injection, dilution, mistake
Filter, sterilizing, to obtain final product.
6. prepare the method for rat compound anesthetic described in claim 2 for one kind, it is characterised in that:
Dexmedetomidine, midazolam, oxycodone, atropine, buffer are dissolved in water for injection,
Dilution, filters, sterilizing, to obtain final product.
7. the rat described in claim 1 or 2 with compound anesthetic in preparing anesthetized rat medicine
Purposes.
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