CN104928350B - Nine kinds of external depression effect rapid screening methods of people's liver CYP450 enzymes - Google Patents
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Abstract
The invention discloses the rapid screening method of 14 kinds of probe substrates of application and 16 kinds of probe reaction comprehensive assessments, 9 kinds of external depression effects of people's liver CYP450 metabolic enzymes.It is quick comprehensively to assess depression effect of the test-compound to metabolic enzyme the invention mainly relates to the metabolic activity change that method and LC/MS/MS combination 9 kinds of people's liver CYP450 enzymes of monitoring are incubated using external mixed probe.This method has considered the selectivity and diversity of probe substrate, influencing each other between probe substrate, temperature incubates different incubation conditions (organic solvents in system, buffer solution, BSA addition etc.) to the activity influence of probe reaction, and every factor such as the enzyme kinetics feature of 16 kinds of probe reactions under selected incubation conditions, and combine high sensitivity, the LC MS/MS technologies of high selectivity, establish a kind of brand-new evaluating in vitro system, to more accurate can more fully predict test-compound to 9 kinds of issuable depression effects of people's liver main metabolic enzyme in the high flux screening of new drug development, improve the foresight of later stage metabolic interaction.
Description
Technical field
The present invention relates to the method that the external depression effect of people's liver CYP450 enzymes is quickly screened, and in particular to is visited using specificity
Pin substrate characterizes enzymatic activity, and a variety of spies are determined simultaneously using external mixed probe substrate method (N-in-one) and LC-MS/MS combinations
Different in nature metabolite, and then differentiate nine kinds of people's liver CYP450 Enzyme activities, and in this, as In vitro metabolism enzyme level effect
Judging basis.
Background technology
For a long time, in Drug safety assessment cytochrome P450s monooxygenase (CYP450) in drug metabolism and medicine
Metabolic suppresses to occupy critical role in interaction.It is pre- as early as possible using experiment in vitro data in the early stage of new drug development
New chemical entities (NCEs) potential drug interaction in vivo is surveyed, obtains associated metabolic information, can be effectively improved new
The success rate of medicine screening, reduces development cost.
External mixed probe substrate method (N-in-one) is combined in new drug development early stage Fast Evaluation with LC-MS/MS methods
NCEs suppresses to be widely applied in interaction to CYP450.Mixed probe substrate method, also referred to as Cocktail probe substrates
Method, i.e., once two or more substrate being metabolized through different enzymes is given in experiment, while obtains multiple enzymatic activity information, in addition
Highly sensitive, high selectivity LC/MS/MS technologies, realize to being detected while a variety of spy medicine metabolins, i.e., monitor simultaneously multiple
CYP450 hypotypes enzyme activity changes, and so as to improve screening flux, improves efficiency, reduces cost.Used in new drug development early stage
There is dispute in N-in-one technologies, reason is to need to consider the influence of many factors, including probe substrate using this method always
Select, avoid substrate interphase interaction, the optimization of incubation system co-factor etc..And the method reported at present still suffers from various degree
The defects of, such as ignore the interaction between substrate, the substrate of different subtype is placed in same system and is incubated, so as to cause vacation
The generation of negative findings.
Mixed probe substrate method typically chooses a kind of Specific probe and characterizes enzymatic activity, but CYP3A4 (Arch
BiochemBiophys, 2001,391 (1)：49-55) and CYP2C9 (Nature, 2003,424 (6947)：The Asia such as 464-468)
Type is necessary to select the incoherent substrate of multiple structures to characterize enzymatic activity because multiple binding sites be present.Constantly have in recent years
CYPs isodynamic enzymes are found to have the substrate dependence of depression effect, as CYP2C8 (Drug Metab Dispos, 2011,39
(9)：1546-1554), CYP2C9 (Drug Metab Dispos, 2009,37 (1)：59-65)、CYP2C19(Drug Metab
Dispos, 2008,36 (3)：523-528), CYP2D6 (Drug Metab Dispos, 2012,40 (1)：47-53) etc..Therefore,
In the in-vitro evaluation of Drug inhibition interaction, especially CYP2 and CYP3, two kinds or two or more knots are selected as far as possible
The incoherent probe substrate of structure characterizes metabolic enzyme activity, with the accuracy for improving the reliability of in vitro results and predicting in vivo.
Drug interaction is there may be between different substrates in same incubation system, as CYP2B6 probe substrates peace is non-
Mephenytoin hydroxylation metabolism that his ketone mediates to CYP2C19, the dextromethorphan-O- demethy lations of CYP2D6 mediations have suppression to make
With (Drug Metab Dispos, 2000,28 (10)：1176-1183), CYP1A2 probe substrates phenacetin is in higher concentrations
Orinase hydroxylation metabolism (Drug Metab Lett, 2011,5 (1) of CYP2C9 mediations can be suppressed：17-24).Prompting exists
Carrying out external mixed probe substrate must avoid application simultaneously such dense in the presence of the substrate to interact, or reduction substrate when temperature is incubated altogether
Spend or take grouping strategy.
In recent years, the report of " BSA (bovine serum albumin(BSA)) effect " in terms of vitro enzyme kinetic parameter is corrected is increasingly
It is more, turn into the hot issue of in vitro-in vivo correlation prediction, the external apparent K after being corrected with BSAmValue carries out external-internal phase
Hepatic clearance is closer to medicine measured value (2~5 times) inside obtained by the analysis of closing property.(the J Pharmacol such as Rowland
Exp Ther, 2007,321 (1)：137-147) research find biomembrane in vitro 37 DEG C be incubated during can discharge some suppressions
The long-chain unsaturated fatty acid (such as oleic acid, linoleic acid, arachidonic acid) of CYPs activity processed, and BSA addition can be closed
These unrighted acids coming off from biomembrane during incubation, reverse the K raisedmValue, is commonly called as " BSA effects ".This
Substrate selective and enzyme selectivity also be present in kind " BSA effects ".And it there is no BSA works are included in mixed probe substrate system at present
For the report of co-factor.In addition, the influence of a variety of organic solvents and buffer solution to enzymatic activity is also different.
The content of the invention
The technical problem to be solved in the invention is：Metabolic enzyme activity is characterized from a variety of different probe substrates, and it is comprehensive
Consider the factors such as the influence and substrate interaction of buffer solution, organic solvent, co-factor to enzymatic activity, optimize mixed probe substrate
Incubation system, quickly screened for the metabolic enzyme depression effect of new drug development early stage.
To solve the above problems, the present invention provides following technical scheme, including：
Substrate selects：One or more probe substrates are selected to characterize enzymatic activity according to each hypotype enzyme characteristic.
Buffer solution is investigated：Each hypotype enzyme activity of CYP450 in Tris-HCl buffer systems and PBS buffer systems is investigated respectively
Property.
Solvent effect is investigated：It is right when the ratio for investigating organic solvent (methanol, acetonitrile) in incubation system is 0.5% and 1%
The influence of each hypotype enzymatic activity.
BSA effects are investigated：Investigate respectively and add various concentrations BSA (0.5%, 1%, 2%) compared with being not added with BSA, each Asia
The activity change of type enzyme.
Substrate interaction is investigated：It is reported that U.S. that CYP2B6 probe substrates Bupropion suppresses CYP2C19 mediations is fragrant appropriate
English hydroxylation metabolism and the dextromethorphan demethylation metabolism of CYP2D6 mediations.Because Bupropion Km values (67-168 μM) are higher,
Therefore Bupropion (0,5,10 μM) is investigated to CYP2C19 another substrate Omeprazole, CYP2D6 another substrate bufuralol
With the presence or absence of inhibitory action.
Sample incubation, processing and measure：By different subtype Specific probe according to above-mentioned solvent effect, buffer solution,
After BSA effects and substrate interaction etc. carry out packet incubation 20min, ice bath terminating reaction and the precipitation for adding containing the internal standard
Agent, LC-MS/MS is determined after the isometric mixing of each group supernatant is taken after centrifugation.
Beneficial effects of the present invention：
The method established of the present invention has considered interaction between the selection of substrate, substrate, buffer solution, organic
Solvent and some temperature incubate the influence of system co-factor to enzymatic activity etc., it is desirable to reduce external mixed probe substrate method prediction medicine
The false negative incidence of interaction, improve the accuracy of prediction result；And screening compounds are main to nine kinds simultaneously for energy
The depression effect of CYP450 hypotypes, improve screening flux.
Brief description of the drawings
Fig. 1：Activity of enzyme reaction compares in Tris-HCl buffer systems and PBS buffer systems
Fig. 2：The influence of organic solvent methanol, acetonitrile to main CYP450 enzymatic activitys
Fig. 3：The metabolism enzyme reaction speed that BSA characterizes on different substrates influences
Fig. 4：The metabolism enzyme reaction speed that Bupropion characterizes on bufuralol and Omeprazole influences
Embodiment
The present invention carries out detailed explanation by the following examples, but is not meant to that present invention is limited only to this.
1 substrate selects
As it was previously stated, mixed probe substrate method, which generally chooses a kind of Specific probe, characterizes enzymatic activity, but for
For CYP3A4 and CYP2C9 etc. has the hypotype of multiple binding sites, the incoherent substrate of multiple structures should be selected to characterize enzyme activity
Property.In addition to multiple binding sites phenomenon, also there is the substrate of depression effect in the hypotype such as CYP2C8, CYP2C9, CYP2C19, CYP2D6
Dependence.Therefore, in the screening technique that the present invention is established, particularly with CYP2C9, CYP2C19, CYP2D6 and CYP3A4, choosing
Select two kinds or the incoherent probe substrate of two or more structures characterizes metabolic enzyme activity (referring to table 1), to improve in vitro results
Reliability and the accuracy predicted in vivo.
1 nine kinds of main CYP450 enzymes hypotypes of table and its selected Specific probe
2 incubated in vitro system optimizations
2.1 experiment material
People's hepatomicrosome (HLM) is purchased from Ruide Liver Disease Inst. (Shanghai) Co., Ltd., and microsomal protein concentration is
10mg/500μl。Na2HPO4·12H2O、NaH2PO4·2H2O、KCl、MgCl2·6H2O is purchased from Nanjing Chemistry Reagent Co., Ltd.,
Tris-base is purchased from Biosharp companies；Nicotinamide-adenine dinucleotide phosphate (NADP+), G6P (G-6-
P), glucose-6-phosphate dehydrogenase (G6PDH), bovine serum albumin(BSA) (BSA), brocasipal (mephenamine) are purchased from
Sigma companies.Methanol (chromatographically pure), acetonitrile (chromatographically pure) are purchased from Merck companies.Specific probe phenacetin
(phenacetin), cumarin (coumarin), taxol (paclitaxel), orinase (tolbutamide), Mei Fen
Appropriate English (S-Mephenytoin), Omeprazole (omeprazole), bufuralol (bufuralol), Chlorzoxazone
(chlorzoxazone), Nifedipine (nifedipine) is purchased from Sigma-Aldrich (St.Louis, MO, USA)；An Feita
Ketone (bupropion) is purchased from Toronto Research Chemicals (Toronto, Canada)；Diclofenac
(diclofenac), dextromethorphan (dextromethorphan) is purchased from damas-beta (Adamas Reagent Co.Ltd)；
Testosterone (testosterone) is purchased from International Laboratory USA；During midazolam (midazolam) is purchased from
State's medicine biological products assay institute.
Phosphate buffer (PBS)：0.1M KCl- phosphate buffers：KCl containing 0.1M, 0.1M Na2HPO4、0.1M
NaH2PO4；PH=7.4.
Tris-HCl buffer solutions：0.1M containing Tris-base, pH=7.5.
NADPH energy-regenerating systems (NRS)：G-6-P containing 10mM, 1.0mM NADP+、2.0U/mL G6PDH
2.2 buffer solutions are investigated
2.2.1 experimental method
Each hypotype enzymatic activitys of CYP450 are investigated in Tris-HCl buffer systems and PBS buffer systems respectively.200 μ l are incubated
System includes：People's hepatomicrosome (final concentration of protein 0.2mgml-1), Specific probe, PBS (pH=7.4) or Tris-
HCl (pH=7.5) buffer solution, MgCl2(10mM)、10mM G-6-P、1.0mM NADP+、2.0U/mL G6PDH；First by substrate with
People's hepatomicrosome preheats 5min in 37 DEG C of water-baths, then adds NRS solution (37 DEG C of water-bath preheatings) and starts reaction, treats 20min
After add 100 μ l containing the internal standards (brocasipal) acetonitrile (0 DEG C of ice bath precooling) terminating reactions and protein precipitation.Then fully shake
3min is swung, 10min protein precipitations are centrifuged with 18000rpm, take supernatant to shift, sample introduction analysis.
2.2.2 experimental result
Buffer runs result is as shown in figure 1, CYP2A6 (coumarin-7-hydroxylation), CYP2C19 (mephenytoins-4 '-hydroxyl
Change), CYP2B6 (Bupropion -2- hydroxylations), CYP2C9 (Diclofenac -4 '-hydroxylation) these four hypotype enzymatic activitys are in Tris-
HCl buffer systems are significantly higher than PBS buffer systems；And CYP3A4 (Omeprazole-S- oxidations), CYP2E1 (Chlorzoxazone -6- hydroxyls
Change) it is then to be significantly higher than Tris-HCl buffer systems in PBS buffer system enzymatic activitys；Remaining metabolic enzyme hypotype is in two kinds of buffer bodies
Enzymatic activity no significant difference in system.Therefore for different enzyme hypotypes, temperature is incubated in experiment and suggested according to each hypotype pair in vitro
The susceptibility of buffer solution chooses suitable buffer solution.
2.3 solvent effects are investigated
2.3.1 experimental method
Organic solvent methanol, acetonitrile are investigated respectively when the ratio during temperature incubates system is 0.5% or 1% to the shadow of enzymatic activity
Ring.200 μ l incubation systems include：People's hepatomicrosome (final concentration of protein 0.2mg.ml-1), Specific probe (methanol or second
Nitrile is prepared, and control organic solvent ratio, control group is with PBS replacements for 0.5% or 1%), PBS (pH=7.4) buffer solution, MgCl2
(10mM)、10mM G-6-P、1.0mM NADP+、2.0U/mL G6PDH；First by substrate and people's hepatomicrosome in 37 DEG C of water-baths
5min is preheated, NRS solution (37 DEG C of water-bath preheatings) is then added and starts reaction, 100 μ l containing the internal standard (adjacent first are added after 20min
Diphenhydramine) acetonitrile (0 DEG C of ice bath precooling) terminating reaction and protein precipitation.Then fully vibration 3min, is centrifuged with 18000rpm
10min protein precipitations, take supernatant to shift, sample introduction analysis.
2.3.2 experimental result
Experimental result as shown in Fig. 2 organic solvent (methanol, acetonitrile) different subtype enzymatic activity is influenceed it is different, methanol and
Acetonitrile (1%) on most of substrate utilization reactivities almost without influence, such as phenacetin, Bupropion, taxol, U.S. fragrant appropriate
The metabolic enzyme activity that English, dextromethorphan, bufuralol, midazolam, testosterone are characterized is almost unchanged.And CYP2E1 (cumarin-
7- is hydroxylated) and CYP2E1 (Chlorzoxazone -6- hydroxylations) to organic solvent then more sensitivity, 1% methanol can significantly reduce CYP2E1
The Chlorzoxazone hydroxylation reaction of mediation is active (33.9%), and the cumarin hydroxylation that 1% acetonitrile can significantly reduce CYP2A6 mediations is anti-
Should activity (40%).Prompt different enzyme hypotypes different and related to organic solvent species to the susceptibility of organic solvent.
2.4 BSA effects
2.4.1 experimental method
It is 0% to investigate concentration of the BSA in system respectively, when 0.5%, 1%, 2%, the influence to enzymatic activity.200 μ l are incubated
The system of educating includes：People's hepatomicrosome (final concentration of protein 0.2mgml-1), Specific probe, PBS (pH=7.4) solution,
MgCl2(10mM), BSA (concentration 0%, 0.5%, 1%, 2%), 10mM G-6-P, 1.0mM NADP+、2.0U/mL G6PDH；First
Substrate and people's hepatomicrosome are preheated into 5min in 37 DEG C of water-baths, NRS solution (37 DEG C of water-bath preheatings) is then added and starts reaction,
100 μ l containing the internal standards (brocasipal) acetonitrile (0 DEG C of ice bath precooling) terminating reactions and protein precipitation are added after 20min.With
Fully vibration 3min afterwards, centrifuges 10min protein precipitations with 18000rpm, takes supernatant to shift, sample introduction analysis.
2.4.2 experimental result
BSA effect experiments result as shown in figure 3, enzyme selectivity and substrate selective be present in influences of the BSA to CYP450,
BSA can accelerate taxol, orinase, bufuralol, dextromethorphan, cumarin, S- mephenytoins, Omeprazole (5- hydroxyls
Change reaction), phenacetin, the metabolic response speed of the substrate such as Bupropion, reduce midazolam, Nifedipine, testosterone, double chlorine
The metabolic response speed of fragrant acid etc., on the metabolic response of Omeprazole (S- oxidations) and Chlorzoxazone then without influence.Wherein Buddhist nun is non-
Horizon oxidative metabolism, the β of testosterone-6-hydroxylation metabolism reaction only reduce its reaction rate when adding high concentration BSA (2%), and right
For Diclofenac hydroxylation metabolism, low concentration BSA (0.5%) can significantly reduce its reaction rate, and this research team speculates
This may be relevant with the high protein Percentage bound of Diclofenac.
2.5 substrate interactions are investigated
2.5.1 experimental method
It is reported that CYP2B6 probe substrates Bupropion suppress CYP2C19 mediation mephenytoin hydroxylation metabolism and
The metabolism of dextromethorphan demethylation (Drug Metab Dispos, 2000,28 (10), the 1176-1183 of CYP2D6 mediations；
Rapid Commun Mass Spectrom, 2005,19 (18), 2651-2658).Due to Bupropion Km values (67-168 μM)
It is higher, therefore Bupropion (0,5,10 μM) is investigated to CYP2C19 another substrate Omeprazole, CYP2D6 another substrate fourth furan
Luo Er whether there is inhibitory action.200 μ l incubation systems include：People's hepatomicrosome (final concentration of protein 0.2mgml-1), An Fei
His ketone (0 μM, 10 μM, 20 μM, 50 μM), bufuralol (5 μM) or Omeprazole (5 μM), PBS (pH=7.4) buffer solution,
MgCl2(10mM), BSA (2%), 10mM G-6-P, 1.0mM NADP+、2.0U/mL G6PDH；First by substrate and people's hepatomicrosome
5min is preheated in 37 DEG C of water-baths, NRS solution (37 DEG C of water-bath preheatings) is then added and starts reaction, 100 μ l are added after 20min
Containing the internal standard (brocasipal) acetonitrile (0 DEG C of ice bath precooling) terminating reaction and protein precipitation.Then fully vibration 3min, with
18000rpm centrifuges 10min protein precipitations, takes supernatant to shift, sample introduction analysis.
2.5.2 experimental result
Experimental result is as shown in figure 4, bufuralol-the 1 '-hydroxylation reaction and CYP2C19 that Bupropion mediates to CYP2D6
Be present different degrees of inhibitory action in the Omeprazole -5- hydroxylation reactions of mediation, to the latter's inhibitory action significantly (Fig. 4), and pacify non-
Also be present stereoselectivity to this inhibitory action of Omeprazole metabolic response in his ketone, i.e., the Aomei of CYP2C19 mediations is drawn
Inhibitory action be present in azoles -5- hydroxylation reactions, and on the Omeprazole-S- oxidations of CYP3A4 mediations without influence.Therefore, in body
Bupropion can not coexist in same incubation system with Omeprazole, bufuralol in outer incubation N-in-one experiments.
3 substrates are grouped and temperature incubates system establishment
3.1 substrates are grouped
Shadow according to the investigation result of above-mentioned substrate interaction and combination buffer, organic solvent, BSA etc. to enzymatic activity
Ring, packet incubation is made to the Specific probe of nine kinds of main CYP450 hypotypes, substrate includes toluene sulphur in wherein Group I
Butyl urea, S- mephenytoins, testosterone and dextromethorphan, substrate includes Nifedipine, cumarin, midazolam, peace in Group II
Non- his ketone and Diclofenac, substrate includes Chlorzoxazone, phenacetin, Omeprazole, taxol and fourth furan Lip river in Group III
You.The principle of the selection gist " in Km values nearby or less than Km values " of concentration of substrate, on the premise of detection sensitivity is ensured, to the greatest extent
Relatively low concentration of substrate may be chosen, avoids producing drug interaction.
3.2 temperature incubate system establishment
According to the influence to enzymatic activity such as above-mentioned buffer solution, organic solvent, BSA, each group incubation system chooses difference respectively
Buffer solution, various concentrations organic solvent and BSA, refer to table 2, so far establish mixed probe substrate temperature and incubate system.
The Specific probe packet of the main CYP450 enzyme hypotypes of 2 nine kinds of table
4 sample incubations, processing and measure
4.1 experiment material
People's hepatomicrosome (HLM) is purchased from Ruide Liver Disease Inst. (Shanghai) Co., Ltd., and microsomal protein concentration is
10mg/500μl。Na2HPO4·12H2O、NaH2PO4·2H2O、KCl、MgCl2·6H2O is purchased from Nanjing Chemistry Reagent Co., Ltd.,
Tris base are purchased from Biosharp；Nicotinamide-adenine dinucleotide phosphate (NADP+), G6P (G-6-P), 6-
Glucose phosphate dehydrogenase (G6PDH), bovine serum albumin(BSA) (BSA), brocasipal (Mephenamine) are purchased from Sigma
Company.Methanol (chromatographically pure), acetonitrile (chromatographically pure) are purchased from Merck companies.Phenacetin (phenacetin), paracetamol
(acetaminophen), cumarin (coumarin), taxol (paclitaxel), orinase (tolbutamide),
4- hydroxy-methylbenzene sulphur butyl ureas (4-hydroxytolbutamide), mephenytoin (S-Mephenytoin), 4 '-hydroxyl are U.S. fragrant appropriate
English (4 '-hydroxymephenytoin), Omeprazole (omeprazole), bufuralol (bufuralol), Chlorzoxazone
(chlorzoxazone), 6- hydroxyls Chlorzoxazone (6-hydroxychlorzoxazone), Nifedipine (nifedipine),
Aoxidize Nifedipine (oxidizednifedipine), 1 '-hydroxyl bufuralol (1 '-hydroxybufuralol), 4 '-hydroxyl
Diclofenac (4 '-hydroxydiclofenac) is purchased from Sigma-Aldrich (St.Louis, MO, USA)；Bupropion
(bupropion), 2- hydroxyls Bupropion (2-hydroxybupropion), 6 Alpha-hydroxy taxols (6 α-
Hydroxypaclitaxel Toronto Research Chemicals (Toronto, Canada)) are purchased from；5- hydroxyls Aomei is drawn
Azoles (5-hydroxyomeprazole) is purchased from J＆K Scientific Ltd；Diclofenac (diclofenac), dextromethorphan
(dextromethorphan) it is purchased from damas-beta (Adamas Reagent Co.Ltd)；Testosterone (testosterone), 6
Beta-hydroxy testosterone (6 β-hydroxytestosterone) is purchased from International Laboratory USA；Midazolam
(midazolam) it is purchased from Nat'l Pharmaceutical ＆ Biological Products Control Institute.1 '-hydroxymidazolam (1 '-hydroxymidazolam),
4 '-hydroxymidazolam (4 '-hydroxymidazolam) is purchased from Fluka.7- Hydroxycoumarins (7-hydroxycoumarin)
Purchased from ChemService S r l. (USA)；Omeprazole sulfone (Omeprazole sulfone) is purchased from TLC PharmaChem
Inc；Dextrorphan (dextrorphan) is purchased from BD Biosciences Discovery Labware (Bedford, USA).Vinegar
Acid, ammonium acetate are purchased from damas-beta (Adamas Reagent Co.Ltd).
Phosphate buffer (PBS)：0.1M KCl- phosphate buffers：KCl containing 0.1M, 0.1M Na2HPO4,0.1M
NaH2PO4；PH=7.4.
Tris-HCl buffer solutions：0.1M containing Tris-base, pH=7.5.
NADPH energy-regenerating systems (NRS)：G-6-P containing 10mM, 1.0mM NADP+、2.0U/mL G6PDH
4.2 sample incubations, processing
200 μ l incubation systems include：50 μ l people hepatomicrosome (final concentration of protein 0.2mgml-1), 1 μ l probe substrates, 1 μ
L inhibitor (NCEs or specific inhibitor), 98 μ l PBS (pH=7.4) or Tris-HCl buffer solutions, 10 μ l MgCl2
(10mM), 10 μ l BSA (0% or 1% or 2%), 30 μ l NRS solution；First by substrate and people's hepatomicrosome in 37 DEG C of thermostatted waters
5min is preheated in bath, NRS solution (37 DEG C of water-bath preheatings) is then added and starts reaction, 100 μ l containing the internal standards are added after 20min
(brocasipal) acetonitrile (0 DEG C of ice bath precooling) terminating reaction and protein precipitation.Then fully vibration 3min, with 18000rpm
10min protein precipitations are centrifuged, takes three groups of Incubating Solutions once to centrifuge after each 100 μ l of supernatant are mixed in equal volume respectively and centrifuges again twice,
Supernatant sample introduction is taken to analyze.
4.3 LC-MS/MS are determined
Instrument：Triple quadrupole bar tandem mass spectrum combined instrument (the supper-fast liquid chromatographic system containing Shimadzu of high performance liquid chromatography one
(UFLC-30AD), the mass spectrometer system of Shimadzu 8050 (Shimadzu, Japan), electric spray ion source and LabSolution LCMS
Ver.5.6 work stations.
Chromatographic condition：Chromatographic column：Phenomenex Luna C18 posts (2.0 × 150mm, 5 μm)；Column temperature：40℃；Flowing
Phase：Aqueous phase (A)：The ultra-pure water of ammonium acetate containing 5mmol/L and 0.01% acetic acid；Organic phase (B)：Methanol：Acetonitrile (1: 1)；Flow velocity：
0.5ml/min, analysis time：10.0min, gradient elution program are as follows：0~0.5min (2%B), 0.5~4.0min (2~
45%B), 4.0~6.5min (45~60%B), 6.5~6.8min (60~80%B), 6.8~7.2min (80~80%B),
7.2~7.5min (80~2%B), 7.5~10.0min (2%B).
Mass Spectrometry Conditions：Positive and negative hypervelocity switches (5msec), and setting source parameter is respectively：Atomization gas flow (Nebulizing
Gaw Flow) 3L/min, heats throughput (Heating Gas Flow) 15L/min, interface temperature (Interface
Temperature) 350 DEG C, 250 DEG C of desolventizing temperature (DL Temperature), heating deblocking temperature (Heat Block
Temperature) 400 DEG C, drier flow (Drying Gas Flow) 5L/min.From multiple ion reaction monitoring (MRM)
Pattern, each Specific probe metabolin MRM parameters see the table below 3.
The present invention establishes the LC-MS/MS methods of the corresponding characteristic metabolic products of each substrate while detection, wherein 4- hydroxyls
Four kinds of metabolins such as base orinase, 4- hydroxyls mephenytoin, 6- hydroxyls Chlorzoxazone and umbelliferone take bear from
Sub- detection pattern, remaining metabolin use positive ion detection pattern.6- hydroxyls Chlorzoxazone, 4- hydroxy-methylbenzene sulphur butyl ureas and 4-
Hydroxyl mephenytoin responds in the negative ion mode is significantly higher than positive ion mode, because this law selects relatively low substrate dense as far as possible
Degree is to reduce the interference of substrate interaction, so the selection negative ion mode detection of these three metabolites.And 7- hydroxyls are fragrant
Although legumin has good mass spectrum to respond under negative ions pattern, the background noise under anionic textiles pattern is far low
In positive ion detection pattern, so umbelliferone also selects anionic textiles pattern.The method that this experiment is established can
Determine the enzymatic activity of nine kinds of CYP450 hypotypes simultaneously, can the quickly screening compounds influence active to CYP450, both time saving height
Effect, and economically feasible.
Each metabolite Mass Spectrometer Method MRM parameters of table 3.
5 nine kinds of external depression effect rapid screening methods of people's liver CYP450 enzymes
Specific inhibitor checking test is carried out to above-mentioned established mixed probe substrate system, by by result and list
Substrate system and existing Reported data compare, and discovery is respectively provided with preferable uniformity, and correlation is good, is shown in Table 4, shows institute
It is reliably feasible to build system.The method being incubated altogether using this mixed probe substrate replaces traditional Single probe substrate prediction CYP450 enzymes
The way of hypotype can be very good the high flux requirement for meeting medicament research and development early stage, be imitated suitable for the suppression to a large amount of drug candidates
Should screen, at the same consider substrate selection, substrate interaction, solvent-susceptible degree, buffer solution and co-factor optimization etc. because
Element, make this mixed probe substrate Forecasting Methodology more reliable, quick convenient detection means is provided for new drug development.
The specific inhibitor of table 4 suppresses IC50 values：Single substrate, mixed probe substrate, literature values
Claims (2)
1. the rapid screening method of nine kinds of people's liver CYP450 enzyme level effects, it is characterised in that：A) methods described can sieve simultaneously
Select nine kinds of main CYP450 enzyme hypotypes, i.e. CYP1A2,2A6,2B6,2C8,2C9,2C19,2D6,2E1,3A4 depression effect；
B) Specific probe selected by each hypotype is：
CYP1A2 Specific probes are phenacetin；
CYP2A6 Specific probes are cumarin；
CYP2B6 Specific probes are Bupropion；
CYP2C8 Specific probes are taxol；
CYP2C9 Specific probes are orinase and Diclofenac；
CYP2C19 Specific probes are Omeprazole and S- mephenytoins；
CYP2D6 Specific probes are bufuralol and dextromethorphan；
CYP2E1 Specific probes are Chlorzoxazone；
CYP3A4 Specific probes are testosterone, midazolam, Nifedipine and Omeprazole；
C) influence and substrate interaction of combination buffer, organic solvent, BSA to enzymatic activity are to selected each hypotype probe substrate
Make packet to be incubated, be grouped as follows shown in table,
A variety of metabolites are detected by LC-MS/MS simultaneously again.
2. the rapid screening method of nine kinds of people liver CYP450 enzyme level effects according to claim 1, it is characterised in that：With
People's hepatomicrosome is that carrier carries out metabolic enzyme depression effect differentiation.
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