CN104055780A - Compound anesthetic for rat and preparation method thereof - Google Patents
Compound anesthetic for rat and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a compound anesthetic for a rat and a preparation method thereof and belongs to the field of preparation and application of animal compound anesthetics. The invention discloses the compound anesthetic for the rat for the first time. The compound anesthetic comprises dexmedetomidine, midazolam, oxycodone, a buffer solution and water for injection. The invention further discloses the preparation method of the compound anesthetic for the rat. The preparation method comprises the following steps: dissolving the above components in the water for injection; diluting; filtering; pilling in an ampoule bottle; and sterilizing to obtain. The compound anesthetic for the rat disclosed by the invention is quick in anesthesia induction, long in anesthesia maintenance time, good in antalgic and sedative effects and stable in revival, and the main influence on physiological and biochemical indexes of the rat is within the physiological range tolerance. The compound anesthetic is simple to operate and can be used for complex or long-term operations of the rat.
Description
Technical field
The present invention relates to a kind of compound anesthetic, relate in particular to compound anesthetic and preparation method thereof for a kind of rat, belong to preparation and the application of muroid animal compound anesthetic.
Background technology
In zoopery, selecting suitable, safe and effective anesthesia is the important foundation of experiment safety and data reliability.Rat is usually used in the research of aspect and the preparations of surgery models such as medicine Nutrition and Metabolism, endocrine, behavioristics, presbyatrics, tumor, infectious disease, cardiovascular disease, and improper a series of pathophysiological change that may cause muroid animal body of anesthesia, even cause its death, affect the result of whole experiment.
At present to have chloral hydrate, urethane, pentobarbital, ketamine, speed to sleep new etc. for domestic conventional rat anesthesia preparation.Chloral hydrate is fugitive anesthetis, and the comparatively safe scope of 10% chloral hydrate is less, and depth of anesthesia is more shallow, of flaccid muscles and analgesic effect is not good, generally can not be for complicated and longer operation process consuming time, while using separately, mortality rate is relatively high, and often needs boost.Urethane is Lente anesthetic agent, when depth of anesthesia reaches surgical operation requirement, to breathing and the obvious inhibitory action of circulation nothing, but this poison of drug is large, there are some researches show that 20% urethane anesthesia can make Rat Erythrocytes aggregation increase, therefore should not, for hemorheological related experiment, confirm that urethane has appreciable impact to the blood of rat, itself has carcinogenecity, thus in the general anesthesia of laboratory animal such anesthetics of recommendation not.Pentobarbital sodium anesthesia is held time about 2h, but larger to the various waveform influences of visual electrophysiology, and lumbar injection has certain inhibitory action to animal body cardiovascular, nervous system simultaneously, and body damage is larger.Ketamine is fugitive anesthetis, and 120mg/kg lumbar injection rat is anaesthetized the only 45min left and right of holding time, narrow application range.Although speed is slept, newly can regulate anesthesia to hold time by controlling dosage, the b wave action to rat retina electrograph in anaesthesia process be larger.
Although the inhalation anesthesia of rat has been realized effective control on depth of anesthesia, while using, need to pass through special-purpose anesthesia equipment, be equipped with single-minded inhalation anesthesia volatilization tank.Not only formality is very loaded down with trivial details, and equipment and medicine very expensive, increase anesthesia cost, most of laboratorys are difficult to apply at home at present.
At present Animal Anesthesia development trend is balanced anesthesia, and multi-medicament use in conjunction namely not only can reasonable control depth of anesthesia, reach the needs of operation, can also reduce single consumption of planting medicine, reduce drug accumulation, alleviate the excessive side effect causing of drug level.Therefore,, for the effect feature of various injecting anesthetic medicines, preparation animal specific compound preparation is the Main Trends of The Development of balanced anesthesia.In addition, because animal has wild nature and non-domestication, operate and loaded down with trivial detailsly cause unavoidably people and animals to damage.Therefore, be badly in need of the compound anesthetic that energy one pin proves effective.And most with prospects be the research of balanced anesthesia compound anesthetic, be new growing point in animal medicine anesthesia field, existing procucts are used for the anaesthesia process of the animals such as dog, deer at present.
Existing rat anesthesia and Baoding still rest on the mutual comparison phase of the use of above-mentioned single medicine, the discussion of dosage and several drugs, need a kind of new compound recipe preparation for callouse badly, solve the existing rat anesthesia agent outstanding problems such as deficiency that aspect exists and the excessive rapid heart rate causing of stress in rats, hyperpiesia of holding time etc. in calmness, analgesia, induction time, anesthesia.
Summary of the invention
One of object of the present invention is to provide a kind of rat compound anesthetic;
Two of object of the present invention is to provide the preparation method of compound anesthetic for a kind of rat.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
First the present invention provides a kind of rat compound anesthetic, comprising: dexmedetomidine, midazolam, oxycodone, buffer and water for injection;
Wherein, described buffer is preferably the phosphate buffer of pH7.0.
First the present invention carries out random combine by the multiple component with anaesthetic effect that is applied to animal, by the contrast test to rat anesthesia, comprehensively analyze the physical signs such as induction of anesthesia phase, anesthetic stage, waking up period, calmness, analgesia, flesh pine effect and breathing, pulse, body temperature of various combinations; By a large amount of screening experiment, found that, the anaesthetic effect of the compound preparation being comprised of dexmedetomidine, midazolam and oxycodone is obviously better than the anaesthetic effect of other various combinations.Therefore, the present invention has determined and has usingd the key component of the compound recipe preparation for callouse that dexmedetomidine, midazolam and oxycodone use as rat.
Dexmedetomidine is a kind of novel high selectivity α
23 adrenergic receptor agonists, it activates nucleus ceruleus α
2receptor, and nucleus ceruleus is α in brain
2the region that A receptor is the most intensive, is the key position of being responsible for reconciling awakening and sleep in brain, thereby produces the non-moving eye sleep of nature (NREM), brings into play calm, angst resistance effect.And dexmedetomidine is slight to respiration inhibition, its metabolism is subject to renal function little, and metabolite does not have activity.Dexmedetomidine has the analgesic activity of moderate, simultaneously its calmness having, reduce stress, with the synergism of opioid drug etc., can reduce the consumption of perioperative opioid drug.The 1 μ g/kg dexmedetomidine that gives fast loading dose, directly activates the α in vascular smooth muscle
2a receptor, produces vasoconstriction, can cause of short duration hypertension, and reflexive ground reduces heart rate.Dexmedetomidine can be deepened rapidly general anesthesia, and anesthesia speed is accelerated, and has its specificity antagonistic, is convenient to regulate the general anesthesia degree of depth, and controllability is better than most general anesthetics.After midazolam induction of anesthesia, each composition of heart rate variability (HRV) all reduces, and the concentration of norepinephrine in blood plasma (NA) obviously reduces, and for its irritability and dexmedetomidine, has good synergism.Quiet note midazolam can cause blood pressure drops, directly suppresses cardiac muscle, expansion peripheral blood vessel and affects autonomic nerve.Dexmedetomidine is also more obvious on the impact of heart rate, blood pressure.The two has synergism in the impact that slows down anesthetic stage laboratory animal heart rate.Oxycodone is the semi-synthetic opioid drug extracting from alkaloid thebaine (Thebaine), the history of applying clinically existing more than 80 year as potent analgesic.Because oxycodone bioavailability is high, route of administration is many, thereby is widely used on clinical medicine, seldom uses this medicine aspect Animal Anesthesia.
Using dexmedetomidine, midazolam and oxycodone as rat on the basis by the key component of compound recipe preparation for callouse having determined, the present invention further optimizes and screens the buffer salt system of the consumption of dexmedetomidine, midazolam and oxycodone and compound anesthetic.
The present invention obtains good 3 consumptions combination of anaesthetic effect to the consumption screening of dexmedetomidine, midazolam and oxycodone, as prescription 1,2 and 3.
Wherein, in prescription 1, each component and content are: described in every 100mL, in compound anesthetic, the content of 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: described in every 100mL, in compound anesthetic, the content of dexmedetomidine 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: described in every 100mL, in compound anesthetic, the content of dexmedetomidine is 15mg, and the content of midazolam is 135mg, and the content of oxycodone is 7mg.
Experimental result finds, although prescription 3 does not have obvious difference at calm and flesh loose measure face and prescription 1 and 2, aspect analgesic effect, prescription 3 is significantly better than prescription 1 and 2.Determine that thus best prescription is prescription 3, described in every 100mL in compound anesthetic, the content of dexmedetomidine is 15mg, and the content of midazolam is 135mg, and the content of oxycodone is 7mg.
Therefore, in order to reach better anaesthetic effect, in compound anesthetic, the content of each component is preferably described in every 100mL: in every 100mL compound anesthetic, and dexmedetomidine 15mg, midazolam 135mg, oxycodone 7mg; Or in every 100mL compound anesthetic, the content of each component is: dexmedetomidine 15mg, midazolam 135mg, oxycodone 6.5mg; Or in every 100mL compound anesthetic, the content of each component is: dexmedetomidine 15mg, midazolam 145mg, oxycodone 7.5mg.
When preparation compound anesthetic, adopt different buffer salt system, there is larger difference in its anaesthetic effect (comprising calmness, flesh pine, analgesia etc.), optimization of the present invention, screened different buffer, final discovery, adopt pH7.0 phosphate buffer preparation anesthetis calm, flesh is loose or the anaesthetic effect such as analgesia on to obviously be better than other buffer.Therefore, the present invention adopts the phosphate buffer preparation compound anesthetic of pH7.0; Wherein, the phosphate buffer of described pH7.0 comprises: 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.
Whether the present invention is also to adding atropine and joining day to test, found that, after injecting atropine, the analgesic effect in anaesthesia process can be strengthened with the key component of compound anesthetic simultaneously, the harmful effect of compound recipe preparation for callouse to rat heart rate and blood pressure can also be effectively improved.The key component of final definite atropine and compound anesthetic adds simultaneously, and dosage is 0.14mg/kg, and in every 100mL compound anesthetic, atropinic content is 14mg.
Atropine belongs to postganglionic anticholinergic drug, and blocking-up muscarinic receptor there are some researches show that the effect that atropine can make nicotine increase heart rate decreases, and its reason may be that atropine has been blocked nicotine cardiac stimulus SNE release acetylcholine.The present invention selects atropine as adjuvant, is because this medicine is atomic little on arterial pressure impact, and the impact of heart rate is also relaxed far beyond epinephrine.It the most significantly acts on is the impact on heart rate, and this effect shows as " biphasic reaction " sometimes, after administration, can occur of short duration decreased heart rate at first, then heart rate speedup.Its accelerating function and original heart rate level are negative correlation, utilize this to be used for relaxing dexmedetomidine heart rate is reduced to amplitude, play the effect of the body that watches for animals.
Wherein, the muroid animal described in the present invention is preferably rat.
The invention provides a kind of method of compound anesthetic for described muroid animal of preparing, the method comprises: dexmedetomidine, midazolam, oxycodone, buffer are dissolved in water for injection, and dilution, filters, and sterilizing, obtains.
The present invention also provides a kind of method of compound anesthetic for described muroid animal of preparing, and the method comprises: dexmedetomidine, midazolam, oxycodone, atropine, buffer are dissolved in water for injection successively to dilution, filter, embedding is in ampoule bottle, and sterilizing, obtains.
Animal of the present invention can be applied to animal especially braking and the anesthesia of rat with compound anesthetic.
Animal of the present invention is compared and has the following advantages with existing Animal Anesthesia agent with compound anesthetic:
1, the induction of anesthesia time is 6.35 ± 2.28min, and it is 130.23 ± 57.22min that anesthesia is held time, and recovery time is 33.28 ± 17.05min.Show that the induction of anesthesia of this compound anesthetic is steady rapidly, the anesthesia length of holding time, revives steadily, without Novel presentation (excited, again sleep etc.).
2, the impact on major physiological parameter: injecting this compound anesthetic has certain influence to normal body temperature, shows as the rear body temperature of injection and starts to decline and continue to maintain low temperature state.Heart rate is compared with basic value during 7.5min after injection, significantly reduces, and uses atropine can effectively alleviate the excessive reduction of heart rate and arrhythmia etc.After respiratory frequency injection, be slight downward trend, one factor analysis of variance difference is not remarkable, lasts till the basic normal level that recovers after anesthesia 120min.Blood oxygen saturation shows as the initial stage and declines, to 20min after injection, be down to minimum, after recover gradually and approach normal level, in whole anaesthesia process, blood oxygen saturation is all more than 93%.Blood pressure starts to raise after injection, to 10min, maintains hypertension state later, reduces gradually blood pressure and 0min diversity all not remarkable (p>0.05) after 80min after 40min.Injecting this compound anesthetic affects within the scope of its physiological tolerance Study on Physiologic.
3, the impact on hepatic and renal function: ALT, AST, ALP, TBIL, BUN, CREA, CHOL etc. reflect the Main Biochemical of hepatic and renal function, and significant change (p>0.05) do not occur in anaesthesia process.
4, simple to operate, lumbar injection, anaesthetizes for rapid induction; Anesthesia is held time longer, and analgesia, sedation effect is good, can be used for the long operation of complexity or time, and recovery time is very fast.
Accompanying drawing explanation
Fig. 1 is heart rate average temporal evolution trend under three kinds of processing; The longitudinal axis represents heart rate (inferior/min), and transverse axis represents time point (min).
Fig. 2 is (the MAP temporal evolution trend of mean arterial pressure under three kinds of different disposal; The longitudinal axis represents blood pressure, and transverse axis represents time point (min).
Fig. 3 is systolic pressure average temporal evolution trend under atropine various dose; The longitudinal axis represents blood pressure, and transverse axis represents time point (min).
The specific embodiment
Below in conjunction with specific embodiment, further describe the present invention, advantage and disadvantage of the present invention will be more clear along with description.It should be understood that described embodiment is only exemplary, does not form any restriction to scope of the present invention.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these modifications or replacement all fall into 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, the content of each component is dexmedetomidine 15mg, midazolam 135mg, and 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: dexmedetomidine, midazolam, oxycodone, potassium dihydrogen phosphate, sodium hydroxide are dissolved in water for injection successively, then add water for injection dilution, filter, embedding, in ampoule bottle, through autoclaving, obtains.
The preparation of embodiment 2 compound anesthetics
In compound anesthetic, each component and content are: in every 100mL compound anesthetic, the content of each component 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: dexmedetomidine, midazolam, oxycodone, atropine, potassium dihydrogen phosphate, sodium hydroxide are dissolved in water for injection successively, then add water for injection dilution, filter, embedding, in ampoule bottle, through autoclaving, obtains.
The preparation of embodiment 3 compound anesthetics
In compound anesthetic, each component and content are: in every 100mL compound anesthetic, the content of each component is dexmedetomidine 15mg, midazolam 135mg, and oxycodone 6.5mg, surplus is water for injection.
The preparation method of compound anesthetic, comprises the following steps: dexmedetomidine, midazolam, oxycodone are dissolved in water for injection successively, then add water for injection dilution, filter, embedding, in ampoule bottle, through autoclaving, obtains.
Laboratory animal
180 of SD rats, the 2-2.5 monthly age, male and female half and half, body weight 258 ± 23g, is provided by Heilongjiang Province university of TCM Experimental Animal Center, raises and test for 4 weeks under identical conditions, and in whole process of the test, feeding and management condition is consistent.
60 of SJL strain white mice, at 1 monthly age, female, body weight 22 ± 3g, is provided by Harbin Medical University's Experimental Animal Center, raises after 2 weeks and test under identical conditions.
4 of white rabbits, at 1 monthly age, male and female are not limit, and body weight is respectively 1.8kg and 2.1kg, by Heilongjiang University of Chinese Medicine's Experimental Animal Center, is provided, and raises after 2 weeks and test under identical conditions.
The component of experimental example 1 compound anesthetic and consumption screening experiment
One, the medicament categories Preliminary screening of compound anesthetic experiment
(1) experimental technique
1. sedation effect is monitored and evaluation criterion
After rat anesthesia, the sedation effect while observing respectively 5min, 10min after medication, 20min and 30min.Observation is blinked, ear is moving, body is moving, head and neck is moving, tail moves.
To sonic stimulation significant reaction, before administration, note "+"; To sonic stimulation bradykinesia, note " ± "; Reactionless to sonic stimulation, note "-".
Methods of marking: "+" meter 0 minute, " ± " meter 1 minute, "-" meter 2 minutes.
2. analgesic effect is monitored and evaluation criterion
Mainly by acupuncture muffle portion, trunk, extremity top, claw, afterbody, evaluate.
Each pain reaction of acupuncture is obvious, note "+"; Pain reaction is blunt, note " ± "; Pain reaction disappears, note "-".
Methods of marking: "+" meter 0 minute, " ± " meter 1 minute, "-" meter 2 minutes.
3. flesh pine effect monitoring and evaluation criterion
Viewing head cervical region, tail, extremity, masseter and abdominal wall muscle relax level.
While mentioning with tweezers folder tail, as appearance is turned one's head and turn round reaction, obviously sliding appears in extremity, stimulation extremity still can be creeped, and remember "+"; There is rotary head in folder tail, extremity slightly slide simultaneously, note " ± " while mentioning; Without turning one's head and turning round reaction, remember "-".
When folder ear is flexible to horizontal level or upwarps as tail while mentioning, note "+"; As there is bending reaction in tail, note " ± "; Tail naturally droops, note "-".
With tweezers, clamp ear and mention, as struggling appears in extremity, note "+"; Extremity slightly slide, note " ± "; Extremity naturally droop, note "-".
When mentioning, folder ear if postabdomen is without significant change, remembers "+"; Postabdomen slightly expands lax, note " ± "; Postabdomen obviously expands lax, note "-".
Masseter is lax gently to be pulled on, lower jaw, and oral cavity can smooth opening, note "+"; Oral cavity can be opened, but very soon can recuperator, note " ± "; Can not open or open rear immediate recovery person, note "-".
Methods of marking: "+" meter 0 minute, " ± " meter 1 minute, "-" meter 2 minutes.
(2) experimental result
The score summation (individual event highest score is 20 * 10 minutes=200 minutes) of all experimental animals in (now anaesthetic effect is comparatively stable) every group when the experimental result in table 1 is 20min.
20min rat anesthesia effect observation (n=20) after table 1 injection
By table 1, first optimize dexmedetomidine and enter next step test as principal agent.
20min rat anesthesia effect observation (n=20) after table 2 injection
The effect that can find out dexmedetomidine+fentanyl and dexmedetomidine+oxycodone by table 2 is best, but comprehensively analyze the factors such as cost, addiction, toxic and side effects and drug control degree of medicine, select dexmedetomidine+oxycodone and enter next step experiment.
20min rat anesthesia effect observation (n=20) after table 3 injection
By table 3, can find out that dexmedetomidine+fentanyl+midazolam effect is best, particularly at ease pain, have better effect, therefore select dexmedetomidine+oxycodone+midazolam as the main component of compound anesthetic.
By the contrast test to rat anesthesia, comprehensive induction of anesthesia phase, anesthetic stage, the waking up period of analyzing various combinations, the physical signs such as calmness, analgesia, flesh pine effect and breathing, pulse, body temperature, the compound preparation that discovery is comprised of dexmedetomidine, midazolam and oxycodone is obviously better than other various combinations, therefore tentatively usings dexmedetomidine, midazolam and the oxycodone main component as compound recipe preparation for callouse.
Two, the usage ratio of each component of compound anesthetic is explored experiment
(1) drug dose Preliminary screening experimental technique
(1) different pharmaceutical ED
50mensuration
Experimental design arranges drug dose by geometric progression, and making adjacent two dosage logarithmic differences is 0.05.Experiment is from estimating that most probable approaches ED
50dosage start, by preliminary experiment, repeatedly explore, discovery may approach ED
50dosage, then each prescription medicinal liquid is diluted to 15mL, in the middle of guaranteeing, dosage, within the scope of 0.010～0.015mL/g, guarantees that ip dosage is within claimed range.In experimentation, according to ground of sequential method principle, carry out, when a rat occur positive reaction (righting reflex loss) next rat just with the dosage of low one-level, on the contrary, when there is negative reaction, use higher leveled dosage.During from first set reaction sign modification, start statistical experiment number of animals (partial results is in Table 4).
(2) different prescription LD
50mensuration
Composition formula LD
50assay method and ED
50mensuration roughly the same, difference is to measure LD
50observed positive index is dead.
Table 4 different pharmaceutical ED
50and LD
50mensuration
(3) anaesthetic effect methods of marking
Specific experiment method is referring to the experimental technique in above-mentioned " the medicament categories Preliminary screening experiment of compound anesthetic ".
(2) experimental result
By the assay method of table 4, determine dexmedetomidine, midazolam and three kinds of medicine ED of oxycodone
50for 0.18mg/kg, 1.00mg/kg, 0.07mg/kg.Using this as drug regimen level 1, increase afterwards by 20% and 40% using dosage, and by Three factors-levels, measure respectively the anaesthetic effect of various combinations, concrete outcome is in Table 5 and table 6.
Table 5 factor level table
(after three kinds of medicine composite injections, 20min's scoring of table 6 white mice anaesthetic effect marks, n=20)
Note: data are relatively takeed on the different capitalizations of mark and represented significant difference (P < 0.05); The identical capitalization of shoulder mark represents difference not remarkable (P > 0.05).
Use the various combination being formed by dexmedetomidine, midazolam and oxycodone to carry out lumbar injection to rat, observe the situations such as biological reflection, calmness, analgesia, flesh pine, the anesthetic action of various combination is comprehensively analyzed and evaluated, Preliminary screening goes out to using dexmedetomidine, midazolam, oxycodone as the component of compound preparation, and to 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 in conjunction with the observation of anaesthetic effect in trial test, design.3 factors in compound recipe preparation for callouse are dexmedetomidine, midazolam, oxycodone, and 3 levels are high, medium and low (table 7).According to orthogonal table L9 (3
3) white mice is divided into 9 test group at random, according to the sample selection principle in statistics standard and pharmacology design, each test group is selected 30 of white mice, and lumbar injection 0.01mL/g, analyzes the impact of each drug component on anaesthetic effect according to orthogonal table.
Table 7 factor level table
The interior righting reflex loss of the rear 30min of white mice anesthesia reaches the above person of 1min and is judged to be the positive.
As shown in Table 8: dexmedetomidine effect when low dosage is better than middle and high dosage; Midazolam effect when low dosage is better than height, middle dosage; Oxycodone effect when middle dosage is better than high and low dose.Comparative analysis experimental result, determines that best of breed is: dexmedetomidine 0.15mg/kg, midazolam 1.35mg/kg, oxycodone 0.07mg/kg.
Table 8 white mice Orthogonal experiment results (n=30)
Note: * represents maximum in every row three level list drug effect fruits, and E1, E2, E3 are the drug effect sum of the basic, normal, high level of this medicine.
The best experiment group 1 of righting reflex effect in option table 8, group 3 are respectively as prescription 1 and prescription 2, by best of breed (dexmedetomidine 0.15mg/kg, midazolam 1.35mg/kg, oxycodone 0.07mg/kg) as prescription 3, enter the decisive confirmatory experiment of next step quadrature.
Four, the decisive confirmatory experiment of quadrature
1 experimental technique
Laboratory animal SD rat is divided into 3 groups at random, i.e. group 1, group 2 and group 3; Every group 20; Group 1, group 2 and group 3 are used respectively by the compound anesthetic 1 of prescription 1 preparation, by the compound anesthetic 2 of prescription 2 preparations, are anaesthetized (consumption of administration, approach and time etc. are identical) with the compound anesthetic 3 of preparing by prescription 3, observe and respectively organize calmness, analgesia, the loose flesh effect after rat anesthesia administration respectively.
Wherein, consisting of of the compound anesthetic 1 that group 1 adopts: described in every 100mL,, in compound anesthetic, 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 adopts: described in every 100mL,, in compound anesthetic, 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 adopts: described in every 100mL,, in compound anesthetic, 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 in Table 9; From the experimental data of table 9, can find out, although the compound anesthetic 3 of preparing by prescription 3 is at calmness and flesh loose measure face and there is no special marked difference by the compound anesthetics of prescription 1 and 2 preparations, but at ease pain, by the compound anesthetic 3 of prescription 3 preparations, will significantly be better than by the compound anesthetic 1 and 2 of prescription 1 and 2 preparations, prescription 3 has exceeded respectively 16.8% and 16.3% than prescription 1 and prescription 2.Determine that thus best prescription is prescription 3.Prescription 3 each components and content are: described in every 100mL, in compound anesthetic, 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 anesthetics
1, experimental technique
The preparation of buffer salt compound anesthetic is divided into groups, is contained in 1.1 experiments
DZ group: sterile saline 1000ml.
JS group (citrate buffer pH6.2): get 2.1% aqueous solution of citric acid, regulate pH value to 6.2 with 50% sodium hydroxide solution, obtain.
Citric acid--sodium hydrogen phosphate buffer (pH4.0).
First liquid: get citric acid 21g or anhydrous citric acid 19.2g and add water and make to be dissolved into 1000ml, put in refrigerator and preserve.
Second liquid: get sodium hydrogen phosphate 71.63g, add water and make to be dissolved into 1000ml.Get above-mentioned first liquid 61.45ml and mix and shake up with second liquid 38.55ml, obtain.
BL group (barbital-sodium chloride pH of buffer 7.8): get barbital sodium 10.1g, add sodium chloride 7.4g and water and dissolve in right amount, separately getting gelatin 1g, to add water appropriate, is incorporated in above-mentioned solution after heating for dissolving.Then with 0.2mol/L hydrochloric acid solution, regulate pH value to 7.8, then be diluted with water to 1000ml, obtain.
LS group (phosphate buffer, pH7.0): get potassium dihydrogen phosphate 6.8g and add 0.1mol/L sodium hydroxide solution 291ml, be diluted with water to 1000ml, obtain.
With water for injection and above-mentioned buffer salt system preparation compound anesthetic.
Each component of compound anesthetic and content are: 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: dexmedetomidine, midazolam, oxycodone are dissolved in buffer salt system successively, and fully vibration mixes, then adds water for injection dilution, filters, and embedding is in ampoule bottle, and autoclaving, both obtained.Keep in Dark Place in 4 ℃.
40 white mice are divided into 4 groups at random, i.e. DZ, LS, JS and BL group, the compound anesthetic 0.01mL/g of every group of lumbar injection respective sets buffer salt system preparation, enters anaesthetic effect monitoring experiment afterwards.
The anaesthetic effect monitoring of 1.2 white mice
1.2.1 sedation effect monitoring
After white mice anesthesia, the sedation effect while observing respectively 5min, 15min after medication, 30min and 60min.Observe nictation, ear is moving, body is moving, head and neck is moving, extremity and tail moving.
1.2.2 analgesic effect monitoring
After white mice anesthesia, the analgesic effect while observing 5min, 15min after medication, 30min and 60min.Mainly by acupuncture muffle portion, trunk, extremity top, claw, afterbody, evaluate.
1.2.3 flesh pine effect monitoring
After white mice anesthesia, the flesh pine effect while observing 5min, 15min after medication, 30min and 60min.Viewing head cervical region, tail, extremity, masseter and abdominal wall muscle relax level.
Sedation effect, analgesic effect and flesh pine effect assessment standard are with the decisive confirmatory experiment of quadrature in experimental example 1.
The interior righting reflex loss of the rear 30min of white mice anesthesia reaches the above person of 1min and is judged to be the positive, according to above standard, evaluates, and with reference to the harmony of calmness, analgesia and flesh pine effect, carries out the Comprehensive Assessment of anaesthetic effect.
1.3 irritation test
1.3.1 intramuscular injection irritation test
Get 4 of healthy rabbits, respectively at a side quadriceps femoris place, inject and respectively to organize water for injection or buffer salt system as the compound anesthetic lmL of solvent preparation, opposite side corresponding site injects equal-volume sterile saline in contrast with method.After 48h, sacrificed by exsanguination white rabbit, takes out quadriceps femoris after dissecting, and longitudinally cuts, and observes the muscle package irritant reaction of injection site, and is converted into corresponding reaction score value by table 4.
The criterion of table 10 intramuscular injection irritation test
1.3.2 eye drip irritation test
Get 4 of healthy rabbits, Baoding, checks two, without abnormal person, can be used for test.Open gently upper and lower eyelid, each group medicinal liquid and each 2 of normal saline are splashed into respectively in images of left and right eyes conjunctival sac, two splash into dose and should equate that the approximately 2～3s of eyelid that then gently sleeps affects result of the test in case medicinal liquid flows out.Observe after both sides ocular administration and normal saline 10,20,30,60,90 and the variation of eye conjunctiva during 120min.
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, can find out, anaesthetizing early stage (5min after injection), anaesthetizing mid-term (15min and 30min after injection), LS group white mice anaesthetic effect is best, calm, flesh is loose, analgesic effect is obviously better than other three groups (P < 0.05).In the anesthesia later stage (60min after injection), calmness, the analgesic effect of LS group are obviously better than other three groups (P < 0.05).
The observation (n=10) of anaesthetic effect after table 11 white mice injection 5min
Note: between group, relatively, the female difference of data shoulder marking-up represents significant differences (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 normal saline injection point zero difference, and injection point meat color is normal, and the reaction score value of 4 rabbit is all cited as 0 minute.The compound anesthetic standard up to specification of different buffer salt system is described, can supplies intramuscular injection.
The demonstration of eye drip irritation test result, after four groups of eye dripping, while only having JS and BL group 10min, an eye conjunctiva presents slight flushing, the tendency of shedding tears.During 25min, bulbar conjunctiva starts to occur flushing, hyperemia, and show slight photophobia, the phenomenon of shedding tears.During to 40min, flushing disappears, and an eye conjunctiva color and luster returns to state before medication substantially, has no that other is abnormal.LS group and contrast DZ group are within the whole observation period, and if unsighted conjunctiva has ANOMALOUS VARIATIONS.
Based on the above results, determine that optimized buffer salt system is that (phosphate buffer pH7.0), is prepared compound anesthetic as buffer system to LS group.
The Anesthetic Effect of experimental example 3 compound anesthetic of the present invention to rat
1, for examination anesthetis
Compound anesthetic comprises: dexmedetomidine, midazolam, oxycodone, potassium dihydrogen phosphate, sodium hydroxide and water for injection; The content of each component is: in every 100mL compound anesthetic, the content of each component is dexmedetomidine 15mg, midazolam 135mg, and 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: dexmedetomidine, midazolam, oxycodone, potassium dihydrogen phosphate, sodium hydroxide are dissolved in water for injection successively, then add water for injection dilution, filter, embedding is in ampoule bottle, and autoclaving, both obtained.
2, experimental technique and result
2.1 anesthesia
Weigh rat body weight.Put into blood pressure holder after it adapts to, by tail cover method, measure its physiological indexes as heart rate (HR), mean arterial pressure (MBP) etc.Rat is put into holder measures its physiological indexes and comprises respiratory frequency (RR), arterial oxygen saturation (SpO after its peace and quiet
2), body temperature (T) etc.Press afterwards 1mL/kg dosage lumbar injection compound anesthetic, monitoring anesthesia period, calmness, analgesia and flesh pine effect.Take heating to measure rat non-invasive blood pressure, connect AD Instruments and lead physiological monitor and MP150 type 16 passage physiological signal instrument more and carry out the indexs such as heart rate, ecg wave form, blood oxygen saturation, body temperature, respiratory frequency and monitor continuously, in experimentation, note heating in good time and insulation.
Respectively at different time points, by heart, gather venous blood 0.5mL, place after 1h, the centrifugal 10min of 1500rpm, the separated serum without chyle and haemolysis is managed to EP.AU5800 series automatic clinical chemistry analyzer is measured the Main Biochemical of the reaction hepatic and renal functions such as its glutamate pyruvate transaminase ALT (U/L), glutamic oxaloacetic transaminase, GOT AST (U/L), alkaline phosphatase (U/L), total bilirubin TBIL (mg/L), blood urea nitrogen BUN (mg/L), creatinine CREA (mg/L), T-CHOL CHOL (mg/L).
2.2 the monitoring in the period of anesthesia
The induction of anesthesia phase is 6.35 ± 2.28min, and to hold time be 130.23 ± 57.22min in anesthesia, and recovery time is 33.28 ± 17.05min, revives steadily, without Novel presentation (excited, again sleep etc.).
2.3 anaesthetic effects (analgesia, calm, flesh is loose) monitoring
By the evaluation criterion in the decisive confirmatory experiment of quadrature in experimental example 1, mark, anaesthetic effect must be 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 function monitorings
Injection compound anesthetic has certain influence to rat normal body temperature, shows as the rear body temperature of injection and starts to decline and continue to maintain low temperature state.Heart rate is compared with basic value during 7.5min after injection, significantly reduces.After respiratory frequency injection, be slight downward trend, one factor analysis of variance difference is not remarkable, lasts till the basic normal level that recovers after anesthesia 120min.Blood oxygen saturation shows as the initial stage and declines, to 20min after injection, be down to minimum, after recover gradually and approach normal level, in whole anaesthesia process, blood oxygen saturation is all more than 93%.Blood pressure starts to raise after injection, to 10min, maintain hypertension state later, after 40min, reduce gradually, 15min-80min blood pressure and 0min significant difference (p < 0.05) after anesthesia, diversity all not significantly (p>0.05) after 80min, in Table 16.
After table 16 anesthesia, every physiological parameter of rat changes (n=10)
Note: with 0min comparison, * represents significant difference, P < 0.05.
2.5 hepatic and renal function monitorings
Compound anesthetic is on rats'liver renal function without obvious impact, and concrete outcome is in Table 17.
After table 17 anesthesia, rats'liver renal function index changes (n=10)
Note: with 0h comparison, * represents significant difference, P < 0.05.
Above result shows, the induction of anesthesia of this compound anesthetic is steady rapidly, and the anesthesia length of holding time, revives steadily, without Novel presentation (excited, again sleep etc.).Anaesthetic effect is good, and analgesia, sedation effect is good, on main physical signs impact within the scope of its physiological tolerance, on rats'liver renal function without obvious impact.
Experimental example 4 adds atropine experiment
On the key component of compound recipe preparation for callouse and the basis of ratio-dependent, to whether adding atropine and atropine joining day to test.
Compound anesthetic comprises: dexmedetomidine, midazolam, oxycodone, potassium dihydrogen phosphate, sodium hydroxide and water for injection; The content of each component is: in every 100mL compound anesthetic, the content of each component is dexmedetomidine 15mg, midazolam 135mg, and oxycodone 7mg, potassium dihydrogen phosphate 680mg, sodium hydroxide 116.4mg, surplus is water for injection.
Get 24 of SD rats and be divided at random tri-groups of A, B, C, press afterwards 1mL/kg dosage lumbar injection compound anesthetic, wherein A group is without atropine; B group is for shifting to an earlier date 15min injecting atropine (0.14mg/kg); C group is and compound anesthetic while injecting atropine (0.14mg/kg).
Experimental result is found, after injecting atropine, can strengthen the analgesic effect (table 18) in anaesthesia process with the key component of compound anesthetic simultaneously, can also effectively improve the harmful effect of compound recipe preparation for callouse to rat heart rate (table 19, table 20, Fig. 1) and blood pressure (table 21, table 22, Fig. 2), especially relax compound anesthetic heart rate is reduced to amplitude.Simultaneously after injecting atropine, induction of anesthesia time and recovery time are without atropine group, 15min injecting atropine group no significant difference (table 23) in advance.The key component of final definite atropine and compound anesthetic adds simultaneously, and dosage is 0.14mg/kg, and, in every 100mL compound anesthetic, atropinic content is 14mg.
From heart rate trend over time, add atropinic heart rate stabilization simultaneously and be better than other two groups, therefore further in the situation that of atropine administration simultaneously, to atropine, add dosage to inquire into.Atropinic consumption and preliminary experiment result while anaesthetizing with reference to dog, test after determining using dosage variable gradient.Result shows that atropine is after medication 15min, and hypertension is (table 24) significantly.
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 three kinds of different disposal of table 20 (
n=8)
Note: * represents with basal heart rate variant, and in table, same letter represents that, under same time conditions, the two difference is not remarkable, different letter representation significant differences, and significant level is 0.05.
The monitoring result of table 21 basic blood pressure (
n=8)
Mean arterial pressure (MAP) monitoring result under three kinds of different disposal of table 22 (
n=8)
Note: * represents with basic blood pressure variant, and in table, same letter represents that, under same time conditions, the two difference is not remarkable, different letter representation significant differences, and significant level is 0.05.
Induction time and recovery time (n=8) under three kinds of different disposal of table 23
Systolic pressure and diastolic pressure average temporal evolution table (n=8) under table 24 atropine various dose
Claims (9)
1. a rat compound anesthetic, is characterized in that, comprising: dexmedetomidine, midazolam, oxycodone, buffer and water for injection.
2. according to rat compound anesthetic claimed in claim 1, it is characterized in that: contain atropine.
3. according to rat compound anesthetic claimed in claim 1, it is characterized in that: the phosphate buffer that described buffer is pH7.0.
4. according to rat compound anesthetic claimed in claim 1, it is characterized in that: in every 100mL compound anesthetic, the content of each component is: dexmedetomidine 15mg, midazolam 135mg, oxycodone 7mg; Or in every 100mL compound anesthetic, the content of each component is: dexmedetomidine 15mg, midazolam 135mg, oxycodone 6.5mg; Or in every 100mL compound anesthetic, the content of each component is: dexmedetomidine 15mg, midazolam 145mg, oxycodone 7.5mg.
5. according to rat compound anesthetic claimed in claim 2, it is characterized in that: in every 100mL compound anesthetic, atropinic content is 14mg.
6. according to rat compound anesthetic claimed in claim 3, it is characterized in that: phosphate buffer comprises potassium dihydrogen phosphate and sodium hydroxide; Wherein, in every 100mL compound anesthetic, the content of potassium dihydrogen phosphate is 680mg, and the content of sodium hydroxide is 116.4mg.
7. prepare described in claim 1 method for compound anesthetic for rat, it is characterized in that: dexmedetomidine, midazolam, oxycodone, buffer are dissolved in water for injection, and dilution, filters, and sterilizing, obtains.
8. prepare described in claim 2 method for compound anesthetic for rat, it is characterized in that: dexmedetomidine, midazolam, oxycodone, atropine, buffer are dissolved in water for injection, and dilution, filters, and sterilizing, obtains.
Rat described in claim 1-6 with compound anesthetic the purposes in preparing anesthetized rat medicine.
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| 李坤等: "右美托咪啶辅助丙泊酚靶控输注在颅内动脉瘤介入栓塞中的麻醉效果", 《解放军医药杂志》, vol. 26, no. 1, 31 January 2014 (2014-01-31), pages 73 - 77 * |
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