CN102650620B - Preparation method, detection method and application of probe drug composition for determination of metabolic activity of cytochrome P450 - Google Patents
Preparation method, detection method and application of probe drug composition for determination of metabolic activity of cytochrome P450 Download PDFInfo
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Abstract
The invention relates to a preparation method, a detection method and application of a probe drug composition for determination of metabolic activity of cytochrome P450. The composition mainly comprises a preparation made with a specific probe with major isoforms of CYP450, i.e. CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4, as an active component. Cocktail probe drug solution is prepared, the probe drug composition is injected into an animal or liver microsomes for in vitro co-incubation, and the concentration of each probe drug is determined to assess the metabolic activity of the CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. In the early stage of research and development of new drugs, the effects of the drugs on the activity of each isoform of the cytochrome P450 are screened in a high-throughput way, and the interactions of the drugs can be predicted. In the stage of clinical research, the testing can be performed with the probe drug composition in an in-vivo probe method, and the effects of the drugs on the in-vivo metabolic activity of different isoforms of the human liver CYP450 can be examined.
Description
Technical field
The present invention relates to field of medicaments, relate to more specifically a kind of " cocktail " probe medicament combination thing of new mensuration Cytochrome P450 metabolic activity, by the variation of its pharmacokinetic parameter evaluate medicine in rat CYP450 subtype C YP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 body, the impact of In vitro metabolism activity.
Background technology
Cytochrome P450 oxidase (cytochrome P450, CYP450) be the topmost enzyme of medicine oxidative metabolism in human body, wherein CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 are topmost hypotypes, content accounts for the more than 80% of CYP450 total amount in liver, and in the medicine of liver metabolism more than 90% by these 6 kinds of hypotype metabolism.
Many factors can affect the activity of CYP450 as inherent cause, age, sex, disease and environment etc., and the activity of CYP450 can affect the metabolic process of medicine, thereby affects the curative effect of medicine; And medicine to CYP450 can produce induction or inhibiting effect, there is drug interaction and bad reaction thereby affect other through the medicine of this enzymes metabolism.Most patients also use at multiple medicines clinically at present, especially the elderly, the situation of simultaneously taking 4～5 kinds of medicines every day is very general, bad reaction due to thing followed drug interaction is also on the rise, and this has become the important and real problem that prescriber and patient must think better of.Drug interaction is generally divided into Pharmacokinetic interaction and the large class of pharmacodynamic interaction two.Pharmacokinetic interaction can occur in absorption, distribution, metabolism, 4 stages of excretion, wherein metabolic interaction incidence is the highest, account for 40% of Pharmacokinetic interaction, clinical meaning is also important, and metabolic interactional 96% is by the mediation of CYP450 enzyme system.At present European and American countries to the determination of activity of CYP450 for new medicament screen and metabolism research, and it is classified as to new drug and declares an experiment must carrying out.In " chemicals Non-clinical Pharmacokinetics investigative technique governing principle " that China promulgates, also actively advocate the activity of CYP450 is studied.
In new medicament screen and discovery stage, to new chemical entities (new chemical entities, NCE) carry out the comprehensive evaluation of pharmacokinetics, the optimum structure of prediction and improvement compound, has become a very important content in new drug research.Good pharmacokinetic parameters and metabolic characteristics are that to have the lead compound of development prospect essential.Therefore, normal by assessing the influence degree of NCE to metabolism in CYP450 specific probe substrate metabolism system in vitro in new drug development process, judge the inhibiting effect of NCE to CYP450, thereby predict the drug interaction that it may cause in vivo, for further clinical drug trial provides valuable information.But external have a great difference with the interior real physiological environment of body, may cause the difference of drug metabolism.Therefore predict drug interaction should body in, in vitro study combines.Composition in the natural products that a large amount of compounds that produce in the face of a large amount of combinatorial chemistry technique and tradition preparation isolation technics obtain, need to adopt the experiment in vivo and vitro method of quick, easy and reliable results and technology to screen for further structure optimization.
At present, be mainly divided in body and the large class of in vitro study two for studying the method for CYP450 activity.The conventional method of in vitro study has hepatomicrosome hatching, genetic recombination P450 enzyme system, hepatocyte cultures and liver tissue slices method.Research report, the result and the clinical manifestation that use the method for the common hatching of specific substrate and hepatomicrosome to measure have good consistance, therefore hepatomicrosome hatching method is more reliable in study in vitro.In vitro study is simple and quick, is applicable to high flux screening.Can also apply as determining the trial test of in vivo studies research direction.But external have a great difference with the interior real physiological environment of body, makes drug metabolism difference to some extent, therefore needs further to carry out research in body.In body, research method mainly contains Genotype and probe medicament method.Genotype is the activity that drug metabolic enzyme is evaluated in the variation of the directly specific DNA of mensuration.But individual metabolic enzyme gene variation occurrence frequency is lower, have not much also to be identified and to be non-characteristic, add genotype detection expense high, not all genotypic variation all causes phenotype change etc. all to limit the practical application of the method.What the most often use is that the method for the activity (phenotype) of directly measuring body endoenzyme is probe medicament method, and probe medicament method is mainly divided into two kinds at present.One is to use separately a kind of probe medicament, even if only select by the catalysis of a kind of specific enzyme hypotype or by several hypotype catalysis but known the material of its metabolic pathway and product thereof, by measuring the metabolic rate in its body, evaluate the active index of this enzyme.But the metabolism of allogenic material often needs plurality of enzymes jointly to participate in, and enzyme has substrate specificity more, and single probe medicament can only reflect the activity of or part CYP450 hypotype, does not have a kind of medicine can evaluate the activity of several hypotypes simultaneously.In practice, often need to understand again the activity of multiple CYP450 hypotypes, so developed the method that gives multiple probe medicaments, i.e. " cocktail " probe medicament method simultaneously.The method can obviously reduce analytical cycle, improves analysis efficiency, the more important thing is that this method can reduce the impact of intraindividual difference on experimental result.This method has obtained widely research in recent years, but the adaptability in drug discovery process also exists certain query to it, wherein mainly pays close attention to that metabolic between each probe medicament interacts and the pharmacodynamic action of probe medicament.Susceptibility and the specificity of cocktail probe medicament method to analytical approach had relatively high expectations simultaneously, will improve like this analysis cost.But there is research to point out under the various requirement prerequisite that meets analytical approach, potential interaction can reduce by the dosage of controlling medicine, and along with the use of present many high specifics and high-sensitivity method, the common method that detects at present most drug plasma concentration has vapor-phase chromatography (GC), liquid phase chromatography (LC) and chromatograph-mass spectrometer coupling method (LC-MS, LC-MS/MS, GC-MS, GC-MS/MS) etc., but these exact instrument are still subject to the restriction of the popularization of testing laboratory's existence conditions and method application.
Cocktail probe medicament method is put forward at the end of the eighties by the people such as Breimer and Schellens, most study be the cocktail solution with 3～5 kinds of probe medicaments composition, use the method for LC/MS/MS to measure the impact of medicine on 3～5 kinds of main CYP450 hypotype activity, specific probe medicine used is different simultaneously.Recently, FDA, EUFEPS and AAPS think in drug metabolism in vivo/interactional research, and the corresponding desirable probe medicament of the each hypotype of CYP450 is respectively: CYP1A2 is choline theophyllinate, caffeine; CYP2C9 is warfarin and orinase; CYP2D6 is desipramine and metoprolol; CYP3A4 is that midazolam and department cut down his spit of fland; CYP2C19 is Omeprazole; CYP2E1 is Chlorzoxazone.
CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 are the topmost six kinds of hypotypes of CYP450, content accounts for the more than 80% of CYP450 total amount in liver, and more than 90% marketed drug is by these 6 kinds of hypotype metabolism.Therefore should contain these six kinds main CYP450 hypotypes for the evaluation of medicine.The cocktail solution of 5 kinds of probe medicaments that comprise caffeine, orinase, metoprolol, Chlorzoxazone and midazolam had once been prepared in previous work by this seminar, and evaluate hepatic ischemia with it and filled with again with IP five kinds of subtype C YP1A2 of rat CYP450, CYP2C9, CYP2D6, the impact (number of patent application: 200810110435.6) of CYP2E1 and CYP3A4 activity.In the method, lacked this important CYP450 hypotype of CYP2C19, and detection method adopted the HPLC method of single wavelength, its recovery is lower, medicine that cannot Accurate Determining low concentration.CYP2C19 and CYP2C9 belong to CYP2C family together, are important CYP450 enzymes, can catalysis many clinical medicines, as Omeprazole, stable, cyclobarbital, Propranolol etc., there is obvious genetic polymorphism simultaneously.This research, on the basis of previous work, has been prepared a kind of new Cytochrome P450 probe medicament combination thing, further optimizes the compatibility condition of probe solution, increases its stability; The accuracy and the precision that use HPLC linear gradient elution method to make the degree of separation of each probe medicament better, improved assay method.For drugs provides a kind of new method to the impact of six kinds of Main Subtype activity of CYP450.
Summary of the invention
In order to address the above problem, the invention provides a kind of probe medicament combination thing of new mensuration CYP450 metabolic activity, study medicine to six Main Subtype functional enzyme CYP1A2 of rat CYP450, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 drug disposition metabolic activity, be intended to set up and evaluate the medicaments sifting model of medicine to CYP450 activity influence, for new drug development, evaluate drug interaction, predict adverse drug reaction, instruct clinical rational drug use that theoretical direction and laboratory foundation are provided.
An object of the present invention is to provide a kind of " cocktail " probe medicament combination thing of six kinds of Main Subtype metabolic activities of CYP450 and preparation method thereof of measuring new time.
Another object of the present invention is to provide high performance liquid chromatography (HPLC) method of simultaneously measuring this probe medicament combination thing.
A further object of the invention be to provide this probe medicament combination thing evaluate medicine on Cytochrome P450 different subtype body in, application in the impact of In vitro metabolism activity.
The probe medicament combination thing of mensuration CYP450 metabolic activity provided by the present invention is to be the preparation that active fraction preparation forms by the specific probe of Main Subtype CYP1A2, the CYP2C9, CYP2C19, CYP2D6, CYP2E1 and the CYP3A4 that contain CYP450.
Wherein, described specific probe is: caffeine, theophylline, warfarin, orinase, Omeprazole, Mephenetoin, metoprolol, dextromethorphan, Chlorzoxazone, dapsone, midazolam or desipramine.
Preferably described specific probe is: caffeine, orinase, Omeprazole, metoprolol, Chlorzoxazone and midazolam.
Weight proportion and the preparation method of the probe medicament combination thing of mensuration CYP450 metabolic activity of the present invention, comprise the following steps:
(1) take 3～10 parts of 10～20 parts of Chlorzoxazones and orinases and be dissolved in absolute ethyl alcohol, for subsequent use;
(2) take 18～35 parts of 3～12 parts of probe medicament caffeines, 18～35 parts of metoprolols, Omeprazole and be dissolved in physiological saline, (1) is added in this solution;
(3) accurately draw again 10～20 parts of midazolams and add above-mentioned mixed liquor, be mixed with " cocktail " probe liquid, make the concentration of each probe medicament be respectively caffeine 0.3～1.2mg/ml, metoprolol 1.8～3.5mg/ml, Omeprazole 1.8～3.5mg/ml, Chlorzoxazone 1～2mg/ml, orinase 0.3～1mg/ml, midazolam 1～2mg/ml.
Caffeine bulk drug (caffeine), Chlorzoxazone bulk drug (chlorzoxazone), metoprolol bulk drug (metoprolol), orinase bulk drug (tolbutamide), Omeprazole bulk drug (omeprazole), midazolam bulk drug described above are preferably midazolam injection (midazolam injection), are commercially available.Chlorzoxazone and orinase are water insoluble, are therefore first dissolved in absolute ethyl alcohol, then add in normal saline solution, to keep good dissolubility.
Fresh configuration is particularly wanted in the probe medicament combination thing preparation preferred solution agent of mensuration CYP450 metabolic activity of the present invention before administration, after preparing, uses immediately.
The present invention also provides the detection method of simultaneously measuring these six kinds of probe medicament combination things.
Detection method of the present invention is to measure the method for these six kinds of CYP450 probe medicament concentration in blood plasma or in microsome, comprises the pre-service of sample and measures high performance liquid chromatography (HPLC) method of these six kinds of probe medicaments simultaneously.
The pre-service of wherein said sample has two kinds, and a kind of is the pre-service of the plasma sample that contains six kinds of probe medicaments of CYP450, and a kind of is the pre-service of the hepatomicrosome sample that contains six kinds of probe medicaments of CYP450.
The pre-service concrete grammar of wherein said plasma sample is: get 1 part of the blood plasma that contains six kinds of probe medicaments of CYP450, add 0.5 part of 1～10 μ g/ml diazepam inner mark solution, after vortex vibration, add 10 parts of chloroform recoveries, vortex, centrifugal, taking off a layer organic phase puts in centrifuge tube, and then to add 10 parts of volume ratios be that normal hexane-chloroform mixed liquor of 6～8: 2～4 extracts, vortex, centrifugal, taking off a layer organic phase puts in centrifuge tube, merge twice and get organic phase, be placed in 40 DEG C of water-baths and dry up with nitrogen, residue dissolves with mobile phase.
The pre-service concrete grammar of wherein said hepatomicrosome sample is: get 1 part of the hepatomicrosome that contains six kinds of probe medicaments of CYP450, add 0.5 part through 4 DEG C of 1～10 cooling μ g/ml diazepam inner mark solutions, vortex vibration, to add 2 parts of volume ratios be 1: 1 through 4 DEG C of cooling methyl alcohol-acetonitrile mixed liquors, vortex, centrifugal, get supernatant sample introduction.
The chromatographic condition of the HPLC method of simultaneously measuring six kinds of probe medicament concentration of CYP450 of the present invention is to use the mutually alkyl linked silicagel column of carbon eighteen incompatibilities, adopt linear gradient elution method, mobile phase is: the mixed solution of methyl alcohol, acetonitrile, 0.05mol/L diammonium hydrogen phosphate pH of buffer 3.4, three's volume ratio time to time change in this mixed solution, it is 30: 10: 60 that operation starts to 5min, 5～8min is 35: 13: 52,8～10min is 35: 14: 51,10～15min is 40: 15: 45,15～35min is 45: 15: 40, and 35～40min is 30: 10: 60.Flow velocity 1.0ml/min, detects wavelength 230nm, 35 DEG C of column temperatures.
Liver microsomes mixed function oxidase system, be called again the MO system that depends on Cytochrome P450, it is a kind of multienzyme electron transport system, be oxido-reductase system important in body, can catalysis numerous endogenous material is if fatty acid, cholesterol, bile acid and steroid hormone and exogenous material are as the metabolism of medicine.What wherein relate to most drug metabolism in body mainly contains 3 gene families (CYP1, CYP2, CYP3).Wherein CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 are topmost hypotypes, content accounts for the more than 80% of CYP450 enzyme total amount in liver, and in the medicine of liver metabolism more than 90% by these 6 kinds of hypotype metabolism.
CYP1A2: in mankind CYP1 family, found P450 oxidase has CYP1A1, CYP1A2 and CYP1B1.Wherein CYP1A2 and drug metabolism relation are the closest, and it accounts for 13% of the total P450 oxidase of liver content, participates in the metabolism of many medicines, steroid hormone and precarcinogen.In human body, the elimination of caffeine 90% is mediated by CYP1A2, and CYP1A2 accounts for absolute leading role in the metabolic process of caffeine, so caffeine is the optimal probe medicament that CYP1A2 activity in body is measured in first-selected being used safely in.
CYP2C9:CYP2C9 accounts for 20% of P450 content, is only second to CYP3A.The many medicines of different nature of its energy metabolism, and also play a role in the activation of precarcinogen, front poisonous substance and mutagenic agent.The medicine of CYP2C9 metabolism includes but not limited to, orinase, phenytoinum naticum, warfarin, TORA, amitriptyline, Prozac, sulfamethoxazole, testosterone and Losartan etc.Preferably phenytoinum naticum, warfarin, orinase are as the medicine of CYP2C9 metabolism.The best is orinase, because orinase in human body almost only with single channel metabolism, methyl hydroxylation forms hydroxy-methylbenzene sulphur butyl urea, is carboxyl orinase subsequently by alcohols and aldehydes dehydrogenase oxidoreductase hydroxy-methylbenzene sulphur butyl urea, and wherein first step reaction is rate-limiting step.This approach can be removed nearly and reach 80%～100% orinase in human body, and in urine, orinase mainly exists with the form of carboxylated product.
CYP2D19:CYP2C19 participates in the metabolism of some important drugs, comprises beta-blocker, antidepressants, proton pump inhibitor etc.Its metabolic drug includes but not limited to mephenytoin, phenytoinum naticum, chloroquine, Omeprazole.Preferably Omeprazole is as the probe medicament of CYP2C19.Omeprazole generates 5-5-Hydroxyomeprazole through CYP2C19 metabolism.
CYP2D6:CYP2D6 participates in the metabolism of clinical over one hundred kind of important drugs, comprises multiple antiarrhymic, beta-blocker, antihypertensive, antidepressants and antipsychotic drug etc.Metabolic drug includes but not limited to debrisoquine, sparteine, dextromethorphan and metoprolol etc., and preferably metoprolol is as the probe medicament of CYP2D6.Metoprolol is mainly through two approach metabolism: the metabolism of O-demethyl and α-hydroxylation metabolism.Wherein α-hydroxylation metabolism generates Alpha-hydroxy metoprolol, is almost formed by the metabolism of CYP2D6 single channel, and therefore metoprolol is the specific substrate of CYP2D6, can be as the probe medicament of this enzymatic activity of research.
CYP2E1: in CYP2E subfamily, CYP2E1 is relatively important, and its substrate is more, wherein major part is precarcinogen and front poisonous substance, small part is medicine.Chlorzoxazone is 6-hydroxyl Chlorzoxazone through CYP2E1 metabolism in vivo, then is combined and is excreted by urine with glucuronic acid.Preferably Chlorzoxazone is as the probe medicament of CYP2E1.
CYP3A:CYP3A participates in the oxidative metabolism of 50～60% clinical commonly used drugs.Rational clinical use and the therapeutic evaluation of its substrate of difference in height direct relation of the substrate popularity of CYP3A and activity thereof.Midazolam is fugitive hypnotic sedative agent, and CYP3A4 and CYP3A5 participate in its internal metabolism reaction, and main metabolites is 1 '-hydroxyl midazolam, is secondly 4-hydroxyl midazolam and Isosorbide-5-Nitrae-dihydroxy midazolam.The liver percentage extraction lower (being about 34%) of midazolam.Therefore, preferred midazolam is as CYP3A metabolic drug.
Six kinds of probe medicaments of the present invention can be used as a new combination and detect the activity of six kinds of CYP450 hypotype enzymes simultaneously, and the interaction in metabolic process between probe medicament can not occur.
The present invention also provide this probe medicament combination thing drugs on CYP450 different subtype body in, purposes aspect the impact of In vitro metabolism activity.
Many factors can affect the activity of CYP450 as inherent cause, age, sex, disease and environment etc., and the activity of CYP450 can affect the metabolic process of medicine, thereby affects the curative effect of medicine; And medicine to CYP450 can produce induction or inhibiting effect, there is drug interaction and bad reaction thereby affect other through the medicine of this enzymes metabolism.Elementary at new drug development, can apply this probe medicament combination thing and carry out liver microsomes incubation experiment, obtain the impact of medicine on external liver CYP450 activity high flux, also can apply this probe medicament combination thing and carry out zoopery, obtain the impact of medicine on Animal Liver CYP450 internal metabolism activity, so as consider pharmacologically active and metabolic enzyme is suppressed and the basis such as induction on to filter out pharmacologically active high and be difficult for causing the interactional noval chemical compound of metabolic.In the clinical research stage, can apply this probe medicament combination thing and carry out the test of intracorporeal probe method, investigate the impact of medicine on people liver CYP450 different subtype internal metabolism activity.
Obviously,, according to foregoing of the present invention, according to ordinary skill knowledge and the means of this area, not departing under the above-mentioned basic fundamental thought of the present invention prerequisite, can also make amendment, replacement or the change of other various ways.
The embodiment of form by the following examples, is described in further detail foregoing of the present invention again.But this should be interpreted as to the scope of the above-mentioned theme of the present invention only limits to following example.All technology realizing based on foregoing of the present invention all belong to scope of the present invention.
Brief description of the drawings
Fig. 1: six kinds of probe medicaments and interior mark mixed standard solution chromatogram.The retention time of each probe medicament is respectively caffeine 4.123min, Chlorzoxazone 6.352min, orinase 15.148min, Omeprazole 18.087min, midazolam 21.606min, diazepam 26.413min.
Fig. 2: rat blank plasma chromatogram.
Fig. 3: rat blank plasma application of sample extraction chromatography figure.The retention time of each probe medicament is respectively caffeine 4.303min, Chlorzoxazone 6.838min, orinase 15.616min, Omeprazole 18.710min, midazolam 21.710min, diazepam 28.780min.
Fig. 4: plasma sample extraction chromatography figure after rat administration.The retention time of each probe medicament is respectively caffeine 4.252min, Chlorzoxazone 6.751min, orinase 15.589min, Omeprazole 18.407min, midazolam 22.008min, diazepam 25.365min.
Fig. 5: caffeine blood concentration average-time plot.
Fig. 6: orinase blood concentration average-time plot.
Fig. 7: Determination of Omeprazole in Plasma average-time plot.
Fig. 8: metoprolol blood concentration average-time plot.
Fig. 9: Chlorzoxazone blood concentration average-time plot.
Figure 10: midazolam blood concentration average-time plot.
Specific embodiment
Further describe flesh and blood of the present invention below in conjunction with embodiments of the invention, this embodiment does not only limit the present invention for the present invention is described.
Embodiment 1
The preparation of probe medicament combination thing of the present invention:
Accurately take Chlorzoxazone 10mg and orinase 5mg is dissolved in 2ml absolute ethyl alcohol, for subsequent use; Accurately take caffeine 5mg, metoprolol 20mg, Omeprazole 35mg is dissolved in 6ml physiological saline, above-mentioned ethanol solution is added in this normal saline solution, accurately draw again midazolam parenteral solution 2ml (10mg) and add above-mentioned mixed liquor, be mixed with " cocktail " probe liquid.The concentration of each probe medicament is respectively caffeine 0.5mg/ml, metoprolol 2mg/ml, Omeprazole 3.5mg/ml, Chlorzoxazone 1mg/ml, orinase 0.5mg/ml, midazolam 1mg/ml.Solution is fresh preparation before administration, after preparing, uses immediately.
Embodiment 2
The preparation of probe medicament combination thing of the present invention:
Accurately take Chlorzoxazone 10mg and orinase 3mg is dissolved in 2ml absolute ethyl alcohol, for subsequent use; Accurately take caffeine 12mg, metoprolol 35mg, Omeprazole 18mg is dissolved in 6ml physiological saline, above-mentioned ethanol solution is added in this normal saline solution, accurately draw again midazolam parenteral solution 2ml (10mg) and add above-mentioned mixed liquor, be mixed with " cocktail " probe liquid.The concentration of each probe medicament is respectively caffeine 1.2mg/ml, metoprolol 3.5mg/ml, Omeprazole 1.8mg/ml, Chlorzoxazone 1mg/ml, orinase 0.3mg/ml, midazolam 1mg/ml.Solution is fresh preparation before administration, after preparing, uses immediately.
Embodiment 3
The preparation of probe medicament combination thing of the present invention:
Accurately take Chlorzoxazone 20mg and orinase 10mg is dissolved in 2ml absolute ethyl alcohol, for subsequent use; Accurately take caffeine 3mg, metoprolol 18mg, Omeprazole 18mg is dissolved in 5ml physiological saline, above-mentioned ethanol solution is added in this normal saline solution, accurately draw again midazolam parenteral solution 3ml (15mg) and add above-mentioned mixed liquor, be mixed with " cocktail " probe liquid.The concentration of each probe medicament is respectively caffeine 0.3mg/ml, metoprolol 1.8mg/ml, Omeprazole 1.8mg/ml, Chlorzoxazone 2mg/ml, orinase 1mg/ml, midazolam 1.5mg/ml.Solution is fresh preparation before administration, after preparing, uses immediately.
Embodiment 4
The preparation of probe medicament combination thing of the present invention:
Accurately take Chlorzoxazone 12mg and orinase 5mg is dissolved in 1.5ml absolute ethyl alcohol, for subsequent use; Accurately take caffeine 8mg, metoprolol 25mg, Omeprazole 25mg is dissolved in 4.5ml physiological saline, above-mentioned ethanol solution is added in this normal saline solution, accurately draw again midazolam parenteral solution 4ml (20mg) and add above-mentioned mixed liquor, be mixed with " cocktail " probe liquid.The concentration of each probe medicament is respectively caffeine 0.8mg/ml, metoprolol 2.5mg/ml, Omeprazole 2.5mg/ml, Chlorzoxazone 1.2mg/ml, orinase 0.5mg/ml, midazolam 2mg/ml.Solution is fresh preparation before administration, after preparing, uses immediately.
Embodiment 5
The preparation of probe medicament combination thing of the present invention:
Accurately take Chlorzoxazone 10mg and orinase 6mg is dissolved in 1.5ml absolute ethyl alcohol, for subsequent use; Accurately take caffeine 5mg, metoprolol 20mg, Omeprazole 25mg is dissolved in 5ml physiological saline, above-mentioned ethanol solution is added in this normal saline solution, accurately draw again midazolam parenteral solution 3.5ml (17.5mg) mg and enter above-mentioned mixed liquor, be mixed with " cocktail " probe liquid.The concentration of each probe medicament is respectively caffeine 0.5mg/ml, metoprolol 2mg/ml, Omeprazole 2.5mg/ml, Chlorzoxazone 1mg/ml, orinase 0.6mg/ml, midazolam 1.75mg/ml.Solution is fresh preparation before administration, after preparing, uses immediately.
Embodiment 6
The preprocess method of plasma sample of the present invention:
Accurately draw plasma sample 200 μ L, add 4 μ gmL
-1diazepam inner mark solution 100 μ L, vortex vibration 30s, adds 2mL to extract solvent methenyl choloride, after vortex 5min, 3500rmin
-1centrifugal 10min.Take off a layer organic phase and put in centrifuge tube, and then add volume ratio be 7: 3 normal hexane-chloroform mixed liquor 2ml extract, after vortex 5min, 3500rmin
-1centrifugal 10min.Take off a layer organic phase and put in centrifuge tube, merge twice and get organic phase, be placed in 40 DEG C of water-baths and dry up with nitrogen, residue redissolves with 100 μ L mobile phases.
Embodiment 7
The preprocess method of plasma sample of the present invention:
Accurately draw plasma sample 200 μ L, add 10 μ gmL
-1diazepam inner mark solution 100 μ L, vortex vibration 30s, adds 2mL to extract solvent methenyl choloride, after vortex 5min, 3500rmin
-1centrifugal 10min.Take off a layer organic phase and put in centrifuge tube, and then add volume ratio be 6: 4 normal hexane-chloroform mixed liquor 2ml extract, after vortex 5min, 3500rmin
-1centrifugal 10min.Take off a layer organic phase and put in centrifuge tube, merge twice and get organic phase, be placed in 40 DEG C of water-baths and dry up with nitrogen, residue redissolves with 100 μ L mobile phases.
Embodiment 8
The preprocess method of plasma sample of the present invention:
Accurately draw plasma sample 200 μ L, add 1 μ gmL
-1diazepam inner mark solution 100 μ L, vortex vibration 30s, adds 2mL to extract solvent methenyl choloride, after vortex 5min, 3500rmin
-1centrifugal 10min.Take off a layer organic phase and put in centrifuge tube, and then add volume ratio be 8: 2 normal hexane-chloroform mixed liquor 2ml extract, after vortex 5min, 3500rmin
-1centrifugal 10min.Take off a layer organic phase and put in centrifuge tube, merge twice and get organic phase, be placed in 40 DEG C of water-baths and dry up with nitrogen, residue redissolves with 100 μ L mobile phases.
Embodiment 9
The preprocess method of hepatomicrosome sample of the present invention:
Get 200 μ l samples, add the 5 μ gmLs cooling through 4 DEG C
-1diazepam inner mark solution 100 μ L, vortex vibration 30s, methyl alcohol-acetonitrile solution 400 μ l protein precipitations that to add through 4 DEG C of cooling volume ratios be 1: 1, vortex 1min, the centrifugal 10min of 12000r/min, gets supernatant 20 μ l sample introductions.
Embodiment 10
The preprocess method of hepatomicrosome sample of the present invention:
Get 200 μ l samples and add the 1 μ gmL cooling through 4 DEG C
-1diazepam inner mark solution 100 μ L, vortex vibration 30s, methyl alcohol-acetonitrile solution 400 μ l protein precipitations that to add through 4 DEG C of cooling volume ratios be 1: 1, vortex 1min, the centrifugal 10min of 12000r/min, gets supernatant 20 μ l sample introductions.
Embodiment 11
The preprocess method of hepatomicrosome sample of the present invention:
Get 200 μ l samples and add the 10 μ gmLs cooling through 4 DEG C
-1diazepam inner mark solution 100 μ L, vortex vibration 30s, methyl alcohol-acetonitrile solution 400 μ l protein precipitations that to add through 4 DEG C of cooling volume ratios be 1: 1, vortex 1min, the centrifugal 10min of 12000r/min, gets supernatant 20 μ l sample introductions.
Embodiment 12
Measure the chromatographic condition of the HPLC method of six kinds of probe medicament concentration of CYP450:
Use the mutually alkyl linked silicagel column of carbon eighteen incompatibilities, adopt linear gradient elution method, mobile phase is: the mixed solution of methyl alcohol, acetonitrile, 0.05mol/L diammonium hydrogen phosphate pH of buffer 3.4, three's volume ratio time to time change in this mixed solution, it is 30: 10: 60 that operation starts to 5min, 5～8min is 35: 13: 52,8～10min is 35: 14: 51,10～15min is 40: 15: 45, and 15～35min is 45: 15: 40, and 35～40min is 30: 10: 60.Flow velocity 1.0ml/min, detects wavelength 230nm, 35 DEG C of column temperatures.
In order to describe better content disclosed by the invention, below enumerate some examples and further illustrated.
The impact of test example 1 tanshinone IIA sodium sulfonate on six kinds of hypotype internal metabolism activity of CYP450
1 medicine and reagent
Midazolam injection (10mg/2ml/ props up, and Jiangsu Nhwa Pharmaceutical Co., Ltd. produces), Chlorzoxazone, metoprolol, orinase, caffeine, diazepam are marketable material medicine.Tanshinone IIA sodium sulfonate parenteral solution (10mg/2ml/ props up, and biochemical the first pharmaceutcal corporation, Ltd in Shanghai produces).
It is pure that absolute ethyl alcohol, diammonium hydrogen phosphate, phosphoric acid, chloroform, normal hexane are commercially available analysis; Methyl alcohol, acetonitrile are commercially available chromatographically pure.Water is distilled water.
2 animals used as test
Wistar rat, male and female half and half, body weight 200 ± 20g, is provided by radiological study institute of Academy of Military Medicine, PLA Experimental Animal Center, and animal produces conformity certification: ASCSK (Tianjin) 2005-0001.
3 chromatographic columns
Agilent zorbax SB-C
18(250mm × 4.6mm, 5 μ m), Agilent company, the U.S..
4 experimental apparatuss
Agilent 1100 HPLC work systems (Agilent company of the U.S.), CAY-1 type liquid flash mixer (Chang'an, Beijing instrument plant), B600 type low speed autobalancing centrifuge (Bai Yang hydro-extractor factory) K-550-GE eddy mixer (ScientificIndustries Inc, the U.S.), CSF-1B ultrasonic generator (Shanghai Ultrasonic Instrument Factory), PB153-S rigorous analysis balance (METTLER TOLEDO, Switzerland), eppendorf pipettor (Germany).
5 experimental techniques:
The preparation of 5.1 probe medicament solution
Accurately take probe medicament caffeine 5mg, metoprolol 20mg, Omeprazole 20mg is dissolved in 6ml physiological saline, take Chlorzoxazone 10mg and orinase 5mg is dissolved in 2ml absolute ethyl alcohol, absolute ethyl alcohol is slowly added in physiological saline, accurately draw again midazolam parenteral solution 2ml (10mg) and add above-mentioned mixed liquor, be mixed with " cocktail " probe liquid.Solution is fresh preparation before administration, after preparing, uses immediately.
5.2 medications and sample collection
5.2.1 experiment grouping
Wistar rat is divided into three groups: control group, low dose group and high dose group, 18 every group.
5.2.2 medication
5.2.2.1 control group:
Control rats every day 1 time, is total to 14d in tail vein injection saline 0.5ml/100g every day; 15d slowly injects " cocktail " probe medicament solution 0.5ml/100g in tail vein.Dosage is respectively caffeine, orinase 2.5mg/kg, Chlorzoxazone, midazolam 5mg/kg, metoprolol, Omeprazole 10mg/kg.
5.2.2.2 low dose group:
Low dose group rat every day 1 time, is total to 14d in tail vein injection Tanshinone I I A sodium sulfonate parenteral solution 3.6mg/Kg every day; 15d slowly injects " cocktail " probe medicament solution 0.5ml/100g in tail vein.Dosage is respectively caffeine, orinase 2.5mg/kg, Chlorzoxazone, midazolam 5mg/kg, metoprolol, Omeprazole 10mg/kg.
5.2.2.3 high dose group:
High dose group rat every day 1 time, is total to 14d in tail vein injection Tanshinone I I A sodium sulfonate parenteral solution 10.8mg/Kg every day; 15d slowly injects " cocktail " probe medicament solution 0.5ml/100g in tail vein.Dosage is respectively caffeine, orinase 2.5mg/kg, Chlorzoxazone, midazolam 5mg/kg, metoprolol, Omeprazole 10mg/kg.
5.2.3 plasma sample collection
Respectively at before injection cocktail probe medicament solution and after administration 5,10,15,20,30min and 1,2,4,8,12,24h intraocular corner of the eyes venous blood sampling 0.8ml be to heparinize centrifuge tube, the centrifugal 10min of 3500rpm, separated plasma, to be measured in-20 DEG C of Refrigerator stores.
5.3 sample pretreatment
Accurately draw plasma sample 200 μ L, add 4 μ gmL
-1diazepam inner mark solution 100 μ L, vortex vibration 30s, adds 2mL to extract solvent methenyl choloride, mixes after 5min 3500rmin in quick liquid mixer
-1centrifugal 10min.Take off a layer organic phase and put in centrifuge tube, and then add normal hexane-chloroform (7: 3) 2ml to extract, mix after 5min 3500rmin in quick liquid mixer
-1centrifugal 10min.Take off a layer organic phase and put in centrifuge tube, merge twice and get organic phase, be placed in 40 DEG C of water-baths and dry up with nitrogen, residue redissolves with 100 μ L mobile phases, gets 20 μ L sample introductions, records chromatogram.
The foundation of 5.4HPLC detection method
5.4.1 chromatographic condition
Mobile phase: methyl alcohol: acetonitrile: 0.05mol/L diammonium hydrogen phosphate damping fluid (pH=3.4; 30: 10: 60 (0-5min), 35: 13: 52 (5-8min), 35: 14: 51 (8-10min), 40: 15: 45 (10-15min), 45: 15: 40 (15-35min), 30: 10: 60 (35-40min); V/v); Detect wavelength: 230nm; Flow velocity: 1.0ml/min; Column temperature: 35 DEG C; Sample size: 20 μ l.
5.4.2 solution preparation
5.4.2.1 the preparation of diammonium hydrogen phosphate damping fluid: take diammonium hydrogen phosphate 2.64g, dissolve with 400ml distilled water, add 85% phosphatase 11 .4ml, regulating pH value is 3.4.
5.4.2.2 the preparation of diazepam inner mark solution: precision takes diazepam 10mg, with anhydrous alcohol solution and be settled in 10ml volumetric flask, obtains mark storing solution in 1mg/ml, puts in 4 DEG C of refrigerators and preserves.Used time adds absolute ethyl alcohol and is diluted to suitable concentration.
5.4.2.3 each probe medicine solution allocation: the stock solution of preparing respectively caffeine, orinase, Omeprazole, metoprolol, Chlorzoxazone and midazolam that concentration is 1mg/ml, except midazolam water dissolves, remaining is all using methyl alcohol as solvent, 4 DEG C of refrigerations, the used time is diluted with water to suitable concentration.
5.4.3 blood plasma typical curve preparation
The each 40 μ l of caffeine, orinase, Omeprazole, metoprolol, Chlorzoxazone and midazolam working fluid that get respectively variable concentrations mix, under nitrogen, dry up, add rat blank plasma 200 μ l, being prepared into concentration is the mixed probe medicine blood plasma titer of 50,20,10,5,2,1,0.5,0.2,0.1 μ g/ml, three parts of the parallel preparations of each concentration.By " sample pretreatment " lower operation, record peak area.Taking caffeine, orinase, Omeprazole, metoprolol, Chlorzoxazone and midazolam sample and interior mark peak area ratio as horizontal ordinate, drug concentration is ordinate, and drawing standard curve, tries to achieve linear regression equation.
5.5 data processing methods and pharmacokinetic analysis
Use the non-compartment model of DAS2.0 software to calculate the main pharmacokinetic parameter of caffeine, orinase, Omeprazole, metoprolol, Chlorzoxazone and midazolam.
5.6 statistical procedures
Result represents with " means standard deviation " (Mean values ± SD), applies SPSS13.0 software and carries out statistical study, and between organizing relatively, Dunnett ' s inspection is compared between two in employing variance analysis.P < 0.05 represents that there were significant differences.
6 results
The selectivity of 6.1HPLC detection method
Under above-mentioned chromatographic condition, the absorption peak peak shape of six kinds of probe medicaments is good, separates completely, meets the testing requirement of biological specimen.Six kinds of probe medicament standard solution add interior target chromatogram to see accompanying drawing 1, and rat blank plasma chromatogram is shown in accompanying drawing 2, and rat blank plasma adds the chromatogram of six kinds of probe medicament standard items and sees accompanying drawing 3, and rat plasma sample chromatogram is shown in accompanying drawing 4.
The typical curve of 6.2 6 kinds of probe medicaments
The typical curve equation (n=3) of caffeine, orinase, Omeprazole, metoprolol, Chlorzoxazone and midazolam in table 1 blood plasma
Six kinds of probe medicament determination of plasma concentration results of 6.3 each groups
Control group, low dose group and high dose group give after cocktail probe solution, the determination of plasma concentration of caffeine, orinase, Omeprazole, metoprolol, Chlorzoxazone and midazolam the results are shown in Table 2-table 7, and its blood concentration average-time curve is shown in accompanying drawing 5～10.
The each group of table 2 rat caffeine blood concentration (
n=6)
The each group of table 3 rat orinase blood concentration (
n=6)
The each group of table 4 rat Determination of Omeprazole in Plasma (
n=6)
The each group of table 5 rat metoprolol blood concentration (
n=6)
The each group of table 6 rat Chlorzoxazone blood concentration (
n=6)
The each group of table 7 rat midazolam blood concentration (
n=6)
The main pharmacokinetic parameter of six kinds of probe medicaments of 6.4 each groups
Use the non-compartment model of DAS2.0 software to calculate the main pharmacokinetic parameter of caffeine, orinase, Omeprazole, metoprolol, Chlorzoxazone and midazolam in control group, low dose group, high dose group.The results are shown in Table 8～table 13.
Main pharmacokinetic parameters after table 8 rat intravenous injection caffeine (2.5mg/kg) (
n=6)
*with control group comparison, P < 0.05
Main pharmacokinetic parameters after table 9 rat intravenous injection orinase (2.5mg/kg) (
n=6)
Main pharmacokinetic parameters after table 10 rat intravenous injection Omeprazole (10mg/kg) (
n=6)
Main pharmacokinetic parameters after table 11 rat intravenous injection metoprolol (10mg/kg) (
n=6)
Main pharmacokinetic parameters after table 12 rat intravenous injection Chlorzoxazone (5mg/kg) (
n=6)
*with control group comparison, P < 0.05
Main pharmacokinetic parameters after table 13 rat intravenous injection midazolam (5mg/kg) (
n=6)
Table 8 data show, compared with control group, and AUC, the t of low dose group and high dose group caffeine
1/2significantly reduce CL significantly raise (P < 0.05) with MRT.Illustrate that vein gives after rat tanshinone IIA sodium sulfonate parenteral solution 14d, caffeine metabolism is in vivo accelerated, and prompting tanshinone IIA sodium sulfonate parenteral solution can obviously strengthen CYP1A2 activity in rat body.
Table 12 data show, compared with control group, AUC and the MRT of low dose group and high dose group Chlorzoxazone significantly reduce, CL significantly raise (P < 0.05).Illustrate that vein gives after rat tanshinone IIA sodium sulfonate parenteral solution 14d, Chlorzoxazone metabolism is in vivo accelerated, and prompting tanshinone IIA sodium sulfonate parenteral solution can obviously strengthen CYP2E1 activity in rat body.
Table 9,10,11,13 data show, compared with control group, vein gives after rat tanshinone IIA sodium sulfonate parenteral solution 14d, the internal metabolism of orinase, Omeprazole, metoprolol, midazolam does not have significant change, illustrates that tanshinone IIA sodium sulfonate parenteral solution is on the active not impact of the internal metabolism of CYP2C9, CYP2C19, CYP2D6 and CYP3A4.
7 conclusions
CYP1A2 and CYP2E1 in Tanshinone I I A sodium sulfonate Injection in Rats body have inducing action, and the activity of CYP2C9, CYP2C19, CYP2D6 and CYP3A4 is not affected.
Test example 2 impacts of Tanshinone I I A sodium sulfonate on six kinds of hypotype In vitro metabolism activity of CYP450
1 medicine and reagent
Rat liver microsomes, 20mg/ml, 0.5ml/ pipe, NADPH regenerative system (A liquid and B liquid) is all purchased from converging the safe health biotechnology (Beijing) of intelligence company limited.
Midazolam injection (10mg/2ml/ props up, and Jiangsu Nhwa Pharmaceutical Co., Ltd. produces), Chlorzoxazone, metoprolol, orinase, caffeine, diazepam are marketable material medicine.Tanshinone IIA sodium sulfonate parenteral solution (10mg/2ml/ props up, and biochemical the first pharmaceutcal corporation, Ltd in Shanghai produces).
It is pure that absolute ethyl alcohol, diammonium hydrogen phosphate, phosphoric acid are commercially available analysis; Methyl alcohol, acetonitrile are commercially available chromatographically pure.Water is distilled water.
2 experimental apparatuss
Agilent 1100 HPLC work systems (Agilent company of the U.S.), CAY-1 type liquid flash mixer, water-bath constant temperature oscillator, supercentrifuge, K-550-GE eddy mixer, PB153-S rigorous analysis balance, eppendorf pipettor.
3 experimental techniques
The preparation of 3.1 probe medicament solution
Accurately take probe medicament caffeine 3mg, metoprolol 15mg, Omeprazole 15mg is dissolved in 6ml physiological saline, take Chlorzoxazone 5mg and orinase 5mg is dissolved in 3ml absolute ethyl alcohol, absolute ethyl alcohol is slowly added in physiological saline, accurately draw again midazolam parenteral solution 1ml (5mg) and add above-mentioned mixed liquor, be mixed with " cocktail " probe liquid.Solution is fresh preparation before administration, after preparing, uses immediately.
3.2 experiment groupings
Experiment is divided into two groups: control group and experimental group, every group parallel does 6 parts.
3.3 hepatomicrosome incubated in vitro methods
Get A liquid 50 μ l, B liquid 10 μ l, PBS damping fluid 420 μ l, add 5 μ l tanshinone IIA sodium sulfonate parenteral solutions (experimental group) or 5 μ l physiological saline (control group), put in water-bath constant temperature oscillator and hatch 5min in 37 DEG C, add 10 μ l rat liver microsomes suspensions (final concentration of protein is 0.4mg/ml), 37 DEG C of aerobics add 5 μ l cocktail probe medicament solution after hatching 5min again, continue 37 DEG C of aerobics and hatch 30min.
3.4 sample pretreatment
Get 500 μ l samples and add methyl alcohol-acetonitrile (1: 1) the solution 500 μ l protein precipitations that contain interior mark diazepam (8 μ g/ml) cooling through 4 DEG C, vortex 1min, the centrifugal 10min of 12000r/min, gets supernatant 20 μ l sample introductions, records chromatogram.
The foundation of 3.5HPLC detection method
3.5.1 chromatographic condition
Mobile phase: methyl alcohol: acetonitrile: 0.05mol/L diammonium hydrogen phosphate damping fluid (pH=3.4; 30: 10: 60 (0-5min), 35: 13: 52 (5-8min), 35: 14: 51 (8-10min), 40: 15: 45 (10-15min), 45: 15: 40 (15-35min), 30: 10: 60 (35-40min); V/v); Detect wavelength: 230nm; Flow velocity: 1.0ml/min; Column temperature: 35 DEG C; Sample size: 20 μ l.
3.5.2 solution preparation
3.5.2.1 the preparation of diammonium hydrogen phosphate damping fluid: take diammonium hydrogen phosphate 2.64g, dissolve with 400ml distilled water, add 85% phosphatase 11 .4ml, regulating pH value is 3.4.
3.5.2.2 the preparation of diazepam inner mark solution: precision takes diazepam 10mg, with anhydrous alcohol solution and be settled in 10ml volumetric flask, obtains mark storing solution in 1mg/ml, puts in 4 DEG C of refrigerators and preserves.Used time adds absolute ethyl alcohol and is diluted to suitable concentration.
3.5.2.3 each probe medicine solution allocation: the stock solution of preparing respectively caffeine, orinase, Omeprazole, metoprolol, Chlorzoxazone and midazolam that concentration is 1mg/ml, except midazolam water dissolves, remaining is all using methyl alcohol as solvent, 4 DEG C of refrigerations, the used time is diluted with water to suitable concentration.
3.5.3 typical curve preparation
Getting respectively rat liver microsomes 5 μ l adds in 500 μ l PBS damping fluids (pH=7.4), make microsome inactivation through 60 DEG C of heating, add caffeine, metoprolol, Chlorzoxazone, Omeprazole, orinase and the midazolam solution 5 μ l of variable concentrations, being prepared into concentration is the mixed probe pharmaceutical standards liquid of 0.31,0.625,1.25,2.5,5,10,20,40,80 μ mol/L, three parts of the parallel preparations of each concentration.By " sample pretreatment " lower operation, record peak area.Taking caffeine, Chlorzoxazone, orinase, metoprolol, midazolam and Omeprazole sample and interior mark peak area ratio as horizontal ordinate, drug concentration is ordinate, and drawing standard curve, tries to achieve linear regression equation.
4.5 data processing methods and pharmacokinetic analysis
Use the non-compartment model of DAS2.0 software to calculate the main pharmacokinetic parameter of caffeine, metoprolol, Chlorzoxazone, Omeprazole, orinase and midazolam.
4.6 statistical procedures
Result represents with " means standard deviation " (Mean values ± SD), applies SPSS13.0 software and carries out statistical study, and between organizing relatively, Dunnett ' s inspection is compared between two in employing variance analysis.P < 0.05 represents that there were significant differences.
5 results
The typical curve of 5.1 6 kinds of probe medicaments
The typical curve equation (n=3) of table 1 caffeine, metoprolol, Chlorzoxazone, Omeprazole, orinase, midazolam
Six kinds of probe medicament determination of plasma concentration results of 5.2 each groups
The measurement result of control group and six kinds of probe medicaments of experimental group is in table 2.
The determination of drug concentration result (n=6) of table 2 control group and experimental group caffeine, metoprolol, Chlorzoxazone, Omeprazole, orinase and midazolam
*compared with control group, P < 0.05
Data in table 2 show, tanshinone IIA sodium sulfonate can obviously suppress the In vitro metabolism of caffeine, obviously accelerates the In vitro metabolism of Chlorzoxazone, on the metabolism of metoprolol, Omeprazole, orinase and midazolam without impact.
6 conclusions
Tanshinone IIA sodium sulfonate can suppress the In vitro metabolism activity of CYP1A2, and the In vitro metabolism activity of induction CYP2E1, has no significant effect CYP2D6, CYP2C9, CYP2C19 and CYP3A4.
Beneficial effect of the present invention
1, the present invention uses six species specificity probe medicaments: caffeine, orinase, Omeprazole, metoprolol, Chlorzoxazone and midazolam, be prepared into cocktail probe medicament combination thing, and set up simple and easy to do, highly sensitive, the HPLC detection method that selectivity is good, can evaluate medicine to six major function enzyme CYP1A2 of CYP450, CYP2C9, CYP2C19, CYP2D6, in CYP2E1 and CYP3A body, the impact of In vitro metabolism activity, set up and evaluated the screening model of medicine to CYP450 activity influence, for new drug development, prediction drug interaction, instruct clinical rational drug use that technological means is provided, theoretical direction and laboratory foundation.
2, six species specificity probe medicament combination things of the present invention: caffeine, Chlorzoxazone, Omeprazole, orinase, metoprolol and midazolam, its pharmacokinetic parameter can be used as and evaluates medicine is the efficiency index of the drug disposition metabolic activity of six major function enzymes to cytochrome oxidase P450.
Claims (7)
1. measure a probe medicament combination thing for Cytochrome P450 metabolic activity, it is characterized in that by the specific probe of Main Subtype CYP1A2, the CYP2C9, CYP2C19, CYP2D6, CYP2E1 and the CYP3A4 that contain CYP450 be the preparation that active fraction preparation forms; Wherein component and mass fraction are as follows:
CYP1A2 specific probe be active component be caffeine: 3～12 parts;
CYP2C9 specific probe be active component be orinase: 3～10 parts;
CYP2C19 specific probe be active component be Omeprazole: 18～35 parts;
CYP2D6 specific probe be active component be metoprolol: 18～35 parts;
CYP2E1 specific probe be active component be Chlorzoxazone: 10～20 parts;
CYP3A4 specific probe be active component be midazolam; 10～20 parts.
2. the preparation method of composition as claimed in claim 1, is characterized in that step is as follows:
(1) take 3～10 parts of 10～20 parts of Chlorzoxazones and orinases and be dissolved in absolute ethyl alcohol, for subsequent use;
(2) take 18～35 parts of 3～12 parts of probe medicament caffeines, 18～35 parts of metoprolols, Omeprazole and be dissolved in physiological saline, (1) is added in this solution;
(3) accurately draw again 10～20 parts of midazolams and add above-mentioned mixed liquor, be mixed with " cocktail " probe liquid, make the concentration of each probe medicament be respectively caffeine 0.3～1.2mg/ml, metoprolol 1.8～3.5mg/ml, Omeprazole 1.8～3.5mg/ml, Chlorzoxazone 1～2mg/ml, orinase 0.3～1mg/ml, midazolam 1～2mg/ml.
3. the application of the probe medicament combination thing of the mensuration Cytochrome P450 metabolic activity of claim 1, give animal by this probe medicament combination thing or hatch altogether with hepatomicrosome is external, evaluating the metabolic activity of CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 by measuring the concentration of each probe medicament; At the new drug development initial stage, high flux screening medicine is for the impact of the each hypotype activity of Cytochrome P450, thus prediction drug interaction.
4. the detection method of these six kinds of probe medicament combination things of the mensuration of claim 3, it is characterized in that measuring the method for six kinds of CYP450 probe medicament concentration in blood plasma or in hepatomicrosome, comprise the pre-service of sample and measure the efficient liquid-phase chromatography method of these six kinds of probe medicaments simultaneously; The chromatographic condition of measuring the HPLC method of six kinds of probe medicament concentration of CYP450 described time is: use the mutually alkyl linked silicagel column of carbon eighteen incompatibilities, adopt linear gradient elution method, mobile phase is: the mixed solution of methyl alcohol, acetonitrile, 0.05mol/L diammonium hydrogen phosphate pH of buffer 3.4, three's volume ratio time to time change in this mixed solution, it is 30:10:60 that operation starts to 5min, 5～8min is 35:13:52,8～10min is 35:14:51,10～15min is 40:15:45,15～35min is 45:15:40, and 35～40min is 30:10:60; Flow velocity 1.0ml/min, detects wavelength 230nm, 35 DEG C of column temperatures.
5. method as claimed in claim 4, the pre-service that it is characterized in that described sample is the pre-service of the plasma sample that contains six kinds of probe medicaments of CYP450; Or the pre-service of the hepatomicrosome sample that contains six kinds of probe medicaments of CYP450.
6. the method as described in claim 4 or 5, the pre-service concrete grammar that it is characterized in that described plasma sample is: get the blood plasma that contains six kinds of probe medicaments of CYP450, add interior mark diazepam solution 1～10 μ g/ml, after vortex vibration, with chloroform recovery, vortex, centrifugal, taking off a layer organic phase puts in centrifuge tube, and then add volume ratio be 6～8:2～4 normal hexane-chloroform mixed liquor extract, vortex, centrifugal, taking off a layer organic phase puts in centrifuge tube, merge twice and get organic phase, being placed in 40 DEG C of water-baths dries up with nitrogen, residue dissolves with mobile phase.
7. the method as described in claim 4 or 5, the pre-service concrete grammar that it is characterized in that described hepatomicrosome sample is: get the hepatomicrosome that contains six kinds of probe medicaments of CYP450, add interior mark diazepam solution 1～10 μ g/ml, vortex vibration, methyl alcohol-acetonitrile mixed liquor that the volume ratio that adds two volumes is 1:1, vortex, centrifugal, get supernatant sample introduction.
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| CN104195218A (en) * | 2014-09-03 | 2014-12-10 | 广东中西达一新药开发有限公司 | Specific probe substrate composition of cytochrome P450 enzyme and application of specific probe substrate composition |
| CN105784865B (en) * | 2016-03-10 | 2018-04-03 | 贵州省烟草公司毕节市公司 | A kind of HPLC MS/MS methods of Chlorzoxazone in detection animal blood plasma |
| CN105664181A (en) * | 2016-04-07 | 2016-06-15 | 乔海灵 | Application of chlorzoxazone serving as probe drug in determination of metabolic activity of human body to nitrosamine compounds |
| CN106290661B (en) * | 2016-11-11 | 2018-08-03 | 重庆市农业科学院 | A kind of method that high performance liquid chromatography tandem mass spectrometry detects CYP2C9 enzyme activities in earthworm body |
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| CN109374772A (en) * | 2018-11-20 | 2019-02-22 | 大理大学 | A method of evaluation Chinese patent drug influences rat cell cytochrome p 450 different subtype enzymatic activity |
| CN109633007A (en) * | 2018-12-28 | 2019-04-16 | 金华职业技术学院 | Method that is a kind of while measuring in rat plasma five kinds of probe medicament contents and its application in pharmacokinetic |
| CN110018251A (en) * | 2019-04-09 | 2019-07-16 | 合肥科颖医药科技有限公司 | A kind of human plasma metoprolol concentration quantitative analysis method |
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