CN104928350A - Method for rapidly screening in-vitro inhibitory effect of nine human liver CYP450 enzymes - Google Patents
Method for rapidly screening in-vitro inhibitory effect of nine human liver CYP450 enzymes Download PDFInfo
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- Plural Heterocyclic Compounds (AREA)
Abstract
The invention discloses a rapid screening method for comprehensively evaluating the in-vitro inhibitory effect of nine human liver CYP450 metabolic enzymes by utilizing 14 probe substrates and 16 probe reaction. The invention mainly relates to a method for monitoring metabolic activity variation of 9 human liver CYP450 enzymes and rapidly and comprehensively evaluating an inhibitory effect of a tested compound on the metabolic enzyme by adopting an in-vitro mixed probe incubation method and LC/MS/MS. According to the method, the exclusiveness and diversity of the probe substrate, interaction of the probe substrates, influence of different inoculation conditions (by charging organic solvent, buffer solution and BSA) in a warm inoculation system and the enzyme kinetics characteristics of 16 probe reactions under the selected inoculation condition are comprehensively considered, a brand new in-vitro system is established by integrating high-sensitive and high-selective LC-MS/MS technology, so that the inhibitory effect of the tested compounds on the nine human liver main metabolic enzymes can be more accurately and comprehensively predicted in the high-throughput screening of the novel drug development, and the predictability on the interaction of the later metabolism can be improved.
Description
Technical field
The present invention relates to the method for people liver CYP450 enzyme vitro inhibition effect rapid screening, be specifically related to adopt Specific probe to characterize enzymic activity, utilize external mixed probe substrate method (N-in-one) and the multiple specific metabolic product of LC-MS/MS coupling Simultaneously test, and then differentiate nine kinds of people liver CYP450 Enzyme activities, and in this, as the judging basis of In vitro metabolism enzyme level effect.
Background technology
For a long time, in Drug safety assessment, cytochrome P450s monooxygenase (CYP450) occupies critical role in drug metabolism and drug-metabolising suppression interact.Utilize experiment in vitro data to predict the drug interaction that new chemical entities (NCEs) is potential in vivo as early as possible at the commitment of new drug development, obtain associated metabolic information, effectively can improve the success ratio of new medicament screen, reduce cost of development.
External mixed probe substrate method (N-in-one) and the coupling of LC-MS/MS method are widely applied in the early stage Fast Evaluation NCEs of new drug development to suppress CYP450 to interact.Mixed probe substrate method, also Cocktail probe substrate method is claimed, namely once experiment gives two or more substrate through different enzymes metabolism, obtain multiple enzymic activity information, in addition the LC/MS/MS technology of highly sensitive, highly selective simultaneously, achieve and detect while multiple spy medicine metabolite, namely monitor the change alive of multiple CYP450 hypotype enzyme simultaneously, thus improve screening flux, raise the efficiency, reduce costs.Use N-in-one technology to there is dispute at new drug development commitment, reason is to adopt this method to need to consider the impact of many factors always, comprises probe substrate and selects, avoids substrate interphase interaction, the optimization of incubation system cofactor etc.And the method reported at present still exists defect in various degree, as ignored the interaction between substrate, the substrate of different subtype being placed in same system and hatching, thus cause the generation of false negative result.
Mixed probe substrate method is generally chosen a kind of Specific probe and is characterized enzymic activity, but CYP3A4 (Arch BiochemBiophys, 2001,391 (1): 49-55) and CYP2C9 (Nature, 2003,424 (6947): 464-468) etc. because there is multiple binding site in hypotype, therefore be necessary to select the incoherent substrate of multiple structure to characterize enzymic activity.CYPs isozyme is constantly had to be found to have the substrate dependency of retarding effect in recent years, 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 interactional in-vitro evaluation of Drug inhibition, especially CYP2 and CYP3, selects two kinds or the incoherent probe substrate of two or more structure to characterize metabolic enzyme activity as far as possible, to improve the accuracy predicted in the reliability of in vitro results and body.
Drug interaction may be there is between different substrates in same incubation system, Dextromethorphane Hbr-O-the demethy lation of the mephenytoin hydroxylation metabolism mediated CYP2C19 as CYP2B6 probe substrate Bupropion, CYP2D6 mediation has restraining effect (Drug Metab Dispos, 2000,28 (10): 1176-1183), tolbutamide hydroxylation metabolism (the Drug Metab Lett that CYP1A2 probe substrate Phenacetin can suppress CYP2C9 to mediate in higher concentrations, 2011,5 (1): 17-24).Prompting must be avoided applying this type of when carrying out the common temperature of external mixed probe substrate and incubating simultaneously and there is interactional substrate, or reduces concentration of substrate or take grouping strategy.
In recent years, " BSA (bovine serum albumin) effect " report in correction vitro enzyme kinetic parameter gets more and more, and becomes the hot issue of in vitro-in vivo correlation prediction, the external apparent K after correcting with BSA
mvalue to carry out in body that in vitro-in vivo correlation analysis obtains hepatic clearance closer to medicine measured value (2 ~ 5 times).(the J Pharmacol Exp Ther such as Rowland, 2007,321 (1): 137-147) research find microbial film in vitro 37 DEG C hatch in process the long-chain unsaturated fatty acid (as oleic acid, linolic acid, arachidonic acid etc.) that can discharge some and suppress CYPs activity, and adding of BSA can be closed these unsaturated fatty acidss in the process of hatching and come off from biomembranous, reverse the K raised
mvalue, is commonly called as " BSA effect ".Also there is substrate selective and enzyme selectivity in this " BSA effect ".And there is no at present include the report of BSA as cofactor in mixed probe substrate system.In addition, multiple organic solvent and damping fluid also different on the impact of enzymic activity.
Summary of the invention
The technical issues that need to address of the present invention are: select multiple different probe substrate to characterize metabolic enzyme activity, and consider damping fluid, organic solvent, cofactor to factors such as the impact of enzymic activity and substrate interactions, optimize mixed probe substrate incubation system, for the metabolic enzyme retarding effect rapid screening that new drug development is early stage.
For solving the problem, the invention provides following technical scheme, comprising:
Substrate is selected: select one or more probe substrate to characterize enzymic activity according to each hypotype enzyme characteristic.
Damping fluid is investigated: investigate each hypotype enzymic activity of CYP450 in Tris-HCl buffer system and PBS buffer system respectively.
Solvent effect is investigated: when in investigation incubation system, the ratio of organic solvent (methyl alcohol, acetonitrile) is 0.5% and 1%, on the impact of each hypotype enzymic activity.
BSA effect is investigated: investigate respectively and add different concns BSA (0.5%, 1%, 2%) and do not add compared with BSA, the activity change of each hypotype enzyme.
Substrate interaction is investigated: it is reported, CYP2B6 probe substrate Bupropion suppresses the mephenytoin hydroxylation metabolism of CYP2C19 mediation and the Dextromethorphane Hbr demethylation metabolism of CYP2D6 mediation.Because Bupropion Km value (67-168 μM) is higher, therefore whether investigation Bupropion (0,5,10 μMs) exists restraining effect to another substrate omeprazole of CYP2C19, another substrate bufuralol of CYP2D6.
Sample incubation, process and mensuration: different subtype Specific probe is carried out after grouping hatches 20min according to above-mentioned solvent effect, damping fluid, BSA effect and substrate interaction etc., ice bath termination reaction also adds containing interior target precipitation agent, and after getting each group of supernatant equal-volume mixing after centrifugal, LC-MS/MS measures.
Beneficial effect of the present invention:
The method synthesis that the present invention sets up considers interaction between the selection of substrate, substrate, damping fluid, organic solvent and some temperature and incubates the impact etc. of system cofactor on enzymic activity, wish the false negative incidence reducing external mixed probe substrate method prediction drug interaction, improve the accuracy predicted the outcome; And SCREENED COMPOUND to the retarding effect of nine kinds of main CYP450 hypotypes, screening flux can be improve simultaneously.
Accompanying drawing explanation
In Fig. 1: Tris-HCl buffer system and PBS buffer system, activity of enzyme reaction compares
Fig. 2: organic solvent methyl alcohol, acetonitrile are on the impact of main CYP450 enzymic activity
Fig. 3: the BSA metabolic enzyme speed of reaction impact that different substrate is characterized
Fig. 4: the metabolic enzyme speed of reaction impact that Bupropion characterizes bufuralol and omeprazole
Embodiment
The present invention carries out detailed explanation by the following examples, but and does not mean that the present invention is only limitted to this.
1 substrate is selected
As previously mentioned, mixed probe substrate method is usually chosen a kind of Specific probe and is characterized enzymic activity, but for the hypotype that CYP3A4 and CYP2C9 etc. exists multiple binding site, the incoherent substrate of multiple structure should be selected to characterize enzymic activity.Except multiple binding sites phenomenon, also there is the substrate dependency of retarding effect in the hypotypes such as CYP2C8, CYP2C9, CYP2C19, CYP2D6.Therefore, in the screening method that the present invention sets up, especially for CYP2C9, CYP2C19, CYP2D6 and CYP3A4, two kinds or the incoherent probe substrate of two or more structure is selected to characterize metabolic enzyme activity (referring to table 1), to improve the accuracy predicted in the reliability of in vitro results and body.
The main CYP450 enzyme hypotype of nine kinds, table 1 and selected Specific probe thereof
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/500 μ l.Na
2hPO
412H
2o, NaH
2pO
42H
2o, KCl, MgCl
26H
2o is purchased from Nanjing Chemistry Reagent Co., Ltd., and Tris-base is purchased from Biosharp company; Triphosphopyridine nucleotide, reduced (NADP+), G6P (G-6-P), glucose-6-phosphate dehydrogenase (G6PDH), bovine serum albumin (BSA), orphenadinum (mephenamine) available from Sigma.Methyl alcohol (chromatographically pure), acetonitrile (chromatographically pure) are purchased from Merck company.Specific probe Phenacetin (phenacetin), tonka bean camphor (coumarin), taxol (paclitaxel), tolbutamide (tolbutamide), mephenytoin (S-Mephenytoin), omeprazole (omeprazole), bufuralol (bufuralol), chlorzoxazone (chlorzoxazone), Nifedipine (ni6edipine) are purchased from Sigma-Aldrich (St.Louis, MO, USA); Bupropion (bupropion) is purchased from Toronto Research Chemicals (Toronto, Canada); Diclofenac (diclofenac), Dextromethorphane Hbr (dextromethorphan) are purchased from damas-beta (Adamas Reagent Co.Ltd); Testosterone (testosterone) is purchased from International Laboratory USA; Midazolam (midazolam) is purchased from Nat'l Pharmaceutical & Biological Products Control Institute.
Phosphate buffered saline buffer (PBS): 0.1M KCl-phosphate buffered saline buffer: containing 0.1M KCl, 0.1M Na
2hPO4,0.1M NaH
2pO4; PH=7.4.
Tris-HCl damping fluid: containing Tris-base 0.1M, pH=7.5.
NADPH energy-regenerating system (NRS): containing 10mM G-6-P, 1.0mM NADP
+, 2.0U/mL G6PDH
2.2 damping fluids are investigated
2.2.1 experimental technique
The each hypotype enzymic activity of CYP450 is investigated respectively in Tris-HCl buffer system and PBS buffer system.200 μ l incubation systems comprise: people's hepatomicrosome (final concentration of protein 0.2mgml
-1), Specific probe, PBS (pH=7.4) or Tris-HCl (pH=7.5) damping fluid, MgCl
2(10mM), 10mM G-6-P, 1.0mM NADP
+, 2.0U/mL G6PDH; The first preheating 5min in 37 DEG C of water-baths by substrate and people's hepatomicrosome, then add NRS solution (37 DEG C of water-bath preheatings) and start reaction, after 20min, add 100 μ l contain interior mark (orphenadinum) acetonitrile (0 DEG C of ice bath precooling) termination reaction and protein precipitation.Fully vibrate 3min subsequently, with the centrifugal 10min protein precipitation of 18000rpm, gets supernatant liquor transfer, sample introduction analysis.
2.2.2 experimental result
As shown in Figure 1, CYP2A6 (coumarin-7-hydroxylation), CYP2C19 (mephenytoin-4 '-hydroxylation), CYP2B6 (Bupropion-2-hydroxylation), CYP2C9 (diclofenac-4 '-hydroxylation) these four kinds of hypotype enzymic activitys are significantly higher than PBS buffer system in Tris-HCl buffer system to buffer runs result; CYP3A4 (omeprazole-S-is oxidized), CYP2E1 (chlorzoxazone-6-hydroxylation) are significantly higher than Tris-HCl buffer system in PBS buffer system enzymic activity; All the other metabolic enzyme hypotypes enzymic activity no significant difference in two kinds of buffer systems.Therefore, for different enzyme hypotype, temperature is incubated in experiment and is advised choosing suitable damping fluid according to the susceptibility of each hypotype to damping fluid in vitro.
2.3 solvent effects are investigated
2.3.1 experimental technique
Investigate organic solvent methyl alcohol, the impact of acetonitrile when the ratio that temperature is incubated in system is 0.5% or 1% on enzymic activity respectively.200 μ l incubation systems comprise: people's hepatomicrosome (final concentration of protein 0.2mgml
-1), Specific probe (methyl alcohol or acetontrile, controlling organic solvent ratio is 0.5% or 1%, and control group replaces with PBS), PBS (pH=7.4) damping fluid, MgCl
2(10mM), 10mM G-6-P, 1.0mM NADP
+, 2.0U/mL G6PDH; The first preheating 5min in 37 DEG C of water-baths by substrate and people's hepatomicrosome, then add NRS solution (37 DEG C of water-bath preheatings) and start reaction, after 20min, add 100 μ l contain interior mark (orphenadinum) acetonitrile (0 DEG C of ice bath precooling) termination reaction and protein precipitation.Fully vibrate 3min subsequently, with the centrifugal 10min protein precipitation of 18000rpm, gets supernatant liquor transfer, sample introduction analysis.
2.3.2 experimental result
Experimental result as shown in Figure 2, organic solvent (methyl alcohol, acetonitrile) is different on the impact of different subtype enzymic activity, methyl alcohol and acetonitrile (1%) on most of substrate utilization reactive behavior almost without impact, as Phenacetin, Bupropion, taxol, mephenytoin, Dextromethorphane Hbr, bufuralol, midazolam, testosterone the metabolic enzyme activity that characterizes almost constant.CYP2E1 (coumarin-7-hydroxylation) and CYP2E1 (chlorzoxazone-6-hydroxylation) is then comparatively responsive to organic solvent, 1% methyl alcohol significantly can reduce the chlorzoxazone hydroxylation reaction activity (33.9%) of CYP2E1 mediation, and 1% acetonitrile significantly can reduce the tonka bean camphor hydroxylation reaction activity (40%) of CYP2A6 mediation.Point out different enzyme hypotype different to the susceptibility of organic solvent, and relevant to organic solvent kind.
2.4BSA effect
2.4.1 experimental technique
Investigating the concentration of BSA in system is respectively 0%, 0.5%, 1%, and when 2%, on the impact of enzymic activity.200 μ l incubation systems comprise: people's hepatomicrosome (final concentration of protein 0.2mgml
-1), Specific probe, PBS (pH=7.4) solution, MgCl
2(10mM), BSA (concentration 0%, 0.5%, 1%, 2%), 10mM G-6-P, 1.0mM NADP
+, 2.0U/mLG6PDH; The first preheating 5min in 37 DEG C of water-baths by substrate and people's hepatomicrosome, then add NRS solution (37 DEG C of water-bath preheatings) and start reaction, after 20min, add 100 μ l contain interior mark (orphenadinum) acetonitrile (0 DEG C of ice bath precooling) termination reaction and protein precipitation.Fully vibrate 3min subsequently, with the centrifugal 10min protein precipitation of 18000rpm, gets supernatant liquor transfer, sample introduction analysis.
2.4.2 experimental result
BSA effect experiment result as shown in Figure 3, there is enzyme selectivity and substrate selective in the impact of BSA on CYP450, BSA can accelerate the metabolic reaction speed of the substrate such as taxol, tolbutamide, bufuralol, Dextromethorphane Hbr, tonka bean camphor, S-mephenytoin, omeprazole (5-hydroxylation reaction), Phenacetin, Bupropion, reduce the metabolic reaction speed of midazolam, Nifedipine, testosterone, diclofenac etc., on the metabolic reaction of omeprazole (S-oxidation) and chlorzoxazone then without affecting.Wherein Nifedipine oxidative metabolism, testosterone-6 β-hydroxylation metabolism reaction only reduce its speed of reaction when adding high density BSA (2%), and for diclofenac hydroxylation metabolism, lower concentration BSA (0.5%) can significantly reduce its speed of reaction, and this research team infers that this may be relevant with the high protein combination rate of diclofenac.
2.5 substrate interactions are investigated
2.5.1 experimental technique
It is reported, CYP2B6 probe substrate Bupropion suppresses the mephenytoin hydroxylation metabolism of CYP2C19 mediation and the metabolism of Dextromethorphane Hbr demethylation (Drug Metab Dispos, 2000,28 (10), 1176-1183 of CYP2D6 mediation; Rapid Commun Mass Spectrom, 2005,19 (18), 2651-2658).Because Bupropion Km value (67-168 μM) is higher, therefore whether investigation Bupropion (0,5,10 μMs) exists restraining effect to another substrate omeprazole of CYP2C19, another substrate bufuralol of CYP2D6.200 μ l incubation systems comprise: people's hepatomicrosome (final concentration of protein 0.2mgml
-1), Bupropion (0 μM, 10 μMs, 20 μMs, 50 μMs), bufuralol (5 μMs) or omeprazole (5 μMs), PBS (pH=7.4) damping fluid, MgCl
2(10mM), BSA (2%), 10mM G-6-P, 1.0mM NADP
+, 2.0U/mL G6PDH; The first preheating 5min in 37 DEG C of water-baths by substrate and people's hepatomicrosome, then add NRS solution (37 DEG C of water-bath preheatings) and start reaction, after 20min, add 100 μ l contain interior mark (orphenadinum) acetonitrile (0 DEG C of ice bath precooling) termination reaction and protein precipitation.Fully vibrate 3min subsequently, with the centrifugal 10min protein precipitation of 18000rpm, gets supernatant liquor transfer, sample introduction analysis.
2.5.2 experimental result
Experimental result as shown in Figure 4, the bufuralol-1 that Bupropion mediates CYP2D6 '-hydroxylation reaction and CYP2C19 mediation omeprazole-5-hydroxylation reaction there is restraining effect in various degree, to the latter's restraining effect significantly (Fig. 4), and also there is stereoselectivity in this restraining effect of Bupropion to omeprazole metabolic reaction, namely to CYP2C19 mediation omeprazole-5-hydroxylation reaction there is restraining effect, and on CYP3A4 mediation omeprazole-S-oxygenizement without impact.Therefore, hatch Bupropion and omeprazole, bufuralol in N-in-one experiment in vitro and can not coexist in same incubation system.
3 substrate groupings and temperature system of incubating are established
3.1 substrate groupings
According to the investigation result of above-mentioned substrate interaction and the impact on enzymic activity such as binding buffer liquid, organic solvent, BSA, the Specific probe of nine kinds of main CYP450 hypotypes is hatched do grouping, wherein in Group I, substrate comprises tolbutamide, S-mephenytoin, testosterone and Dextromethorphane Hbr, in Group II, substrate comprises Nifedipine, tonka bean camphor, midazolam, Bupropion and diclofenac, and in Group III, substrate comprises chlorzoxazone, Phenacetin, omeprazole, taxol and bufuralol.The principle of the selection gist of concentration of substrate " near Km value or be less than Km value ", under the prerequisite ensureing detection sensitivity, chooses lower concentration of substrate as far as possible, avoids producing drug interaction.
3.2 temperature incubate system establishment
According to the impact on enzymic activity such as above-mentioned damping fluid, organic solvent, BSA, each group incubation system chooses different damping fluid, different concns organic solvent and BSA respectively, refers to table 2, so far establishes mixed probe substrate temperature and incubate system.
The Specific probe grouping of the main CYP450 enzyme hypotype of nine kinds, table 2
4 sample incubation, process and mensuration
4.1 experiment material
People's hepatomicrosome (HLM) is purchased from Ruide Liver Disease Inst. (Shanghai) Co., Ltd., and microsomal protein concentration is 10mg/500 μ l.Na
2hPO
412H
2o, NaH
2pO
42H
2o, KCl, MgCl
26H
2o is purchased from Nanjing Chemistry Reagent Co., Ltd., and Tris base is purchased from Biosharp; Triphosphopyridine nucleotide, reduced (NADP+), G6P (G-6-P), glucose-6-phosphate dehydrogenase (G6PDH), bovine serum albumin (BSA), orphenadinum (Mephenamine) available from Sigma.Methyl alcohol (chromatographically pure), acetonitrile (chromatographically pure) are purchased from Merck company.Phenacetin (phenacetin), paracetamol (acetaminophen), tonka bean camphor (coumarin), taxol (paclitaxel), tolbutamide (tolbutamide), 4-hydroxytoluene sulphur butyl urea (4-hydroxytolbutamide), mephenytoin (S-Mephenytoin), 4 '-hydroxyl mephenytoin (4 '-hydroxymephenytoin), omeprazole (omeprazole), bufuralol (bufuralol), chlorzoxazone (chlorzoxazone), 6-hydroxyl chlorzoxazone (6-hydroxychlorzoxazone), Nifedipine (nifedipine), oxidation Nifedipine (oxidizednifedipine), 1 '-hydroxyl bufuralol (1 '-hydroxybufuralol), 4 '-hydroxyl diclofenac (4 '-hydroxydiclofenac) is purchased from Sigma-Aldrich (St.Louis, MO, USA), Bupropion (bupropion), 2-hydroxyl Bupropion (2-hydroxybupropion), 6 Alpha-hydroxy taxols (6 α-hydroxypaclitaxel) are purchased from Toronto Research Chemicals (Toronto, Canada), 5-Hydroxyomeprazole (5-hydroxyomeprazole) is purchased from J & K Scientific Ltd, diclofenac (diclofenac), Dextromethorphane Hbr (dextromethorphan) are purchased from damas-beta (Adamas Reagent Co.Ltd), testosterone (testosterone), 6 beta-hydroxy testosterones (6 β-hydroxytestosterone) are purchased from International Laboratory USA, midazolam (midazolam) is purchased from Nat'l Pharmaceutical & Biological Products Control Institute.1 '-hydroxymidazolam (1 '-hydroxymidazolam), 4 '-hydroxymidazolam (4 '-hydroxymidazolam) purchased from Fluka.7-Hydroxycoumarin (7-hydroxycoumarin) purchased from ChemService Srl. (USA); Omeprazole sulfone (Omeprazole sulfone) is purchased from TLCPharmaChem Inc; Levorphanol d-form (dextrorphan) is purchased from BD Biosciences Discovery Labware (Bedford, USA).Acetic acid, ammonium acetate are purchased from damas-beta (Adamas Reagent Co.Ltd).
Phosphate buffered saline buffer (PBS): 0.1M KCl-phosphate buffered saline buffer: containing 0.1M KCl, 0.1M Na2HPO4,0.1MNaH2PO4; PH=7.4.
Tris-HCl damping fluid: containing Tris-base 0.1M, pH=7.5.
NADPH energy-regenerating system (NRS): containing 10mM G-6-P, 1.0mM NADP
+, 2.0U/mL G6PDH
4.2 sample incubation, process
200 μ l incubation systems comprise: 50 μ l people hepatomicrosome (final concentration of protein 0.2mgml
-1), 1 μ l probe substrate, 1 μ l inhibitor (NCEs or specific inhibitor), 98 μ l PBS (pH=7.4) or Tris-HCl damping fluids, 10 μ l MgCl
2(10mM), 10 μ l BSA (0% or 1% or 2%), 30 μ l NRS solution; The first preheating 5min in 37 DEG C of waters bath with thermostatic control by substrate and people's hepatomicrosome, then add NRS solution (37 DEG C of water-bath preheatings) and start reaction, after 20min, add 100 μ l contain interior mark (orphenadinum) acetonitrile (0 DEG C of ice bath precooling) termination reaction and protein precipitation.Fully vibrate 3min subsequently, with the centrifugal 10min protein precipitation of 18000rpm, gets three groups of Incubating Solutions respectively once after each 100 μ l equal-volumes mixings of centrifugal supernatant more centrifugal twice, get the analysis of supernatant sample introduction.
4.3LC-MS/MS measure
Instrument: high performance liquid chromatography-triple quadrupole bar tandem mass spectrum combined instrument is (containing the supper-fast liquid chromatographic system of Shimadzu (UFLC-30AD), Shimadzu 8050 mass spectrometer system (Shimadzu, Japan), electric spray ion source and LabSolution LCMS Ver.5.6 workstation.
Chromatographic condition: chromatographic column: Phenomenex Luna C18 post (2.0 × 150mm, 5 μm); Column temperature: 40 DEG C; Moving phase: aqueous phase (A): containing the ultrapure water of 5mmol/L ammonium acetate and 0.01% acetic acid; Organic phase (B): methyl alcohol: acetonitrile (1: 1); Flow velocity: 0.5ml/min, analysis time: 10.0min, gradient elution program is 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 electrode switches (5msec) at a high speed, setting source dates is respectively: atomization gas flow (Nebulizing Gaw Flow) 3L/min, heat air flow (Heating Gas Flow) 15L/min, interface temperature (Interface Temperature) 350 DEG C, desolventizing temperature (DL Temperature) 250 DEG C, heat block temperature (Heat Block Temperature) 400 DEG C, moisture eliminator flow (Drying Gas Flow) 5L/min.Select multiple ion reaction monitoring (MRM) pattern, each Specific probe metabolite MRM parameter sees the following form 3.
The present invention establishes the LC-MS/MS method that the corresponding characteristic metabolic products of each substrate detects simultaneously, wherein four kinds of metabolites such as 4-hydroxytoluene sulphur butyl urea, 4-hydroxyl mephenytoin, 6-hydroxyl chlorzoxazone and umbelliferone take anionic textiles pattern, and all the other metabolites all adopt positive ion detecting pattern.6-hydroxyl chlorzoxazone, 4-hydroxytoluene sulphur butyl urea and 4-hydroxyl mephenytoin respond in the negative ion mode and are significantly higher than positive ion mode, because this law selects lower concentration of substrate to reducing the interference of substrate interaction as far as possible, so these three kinds of meta-bolitess select negative ion mode to detect.Although and umbelliferone has good mass spectrum response under negative ions pattern, the background noise under anionic textiles pattern is far below positive ion detecting pattern, so umbelliferone also selects anionic textiles pattern.The method set up of this experiment can the enzymic activity of Simultaneously test nine kinds of CYP450 hypotypes, can rapid screening compound on the impact of CYP450 activity, both time-saving and efficiency, again economically feasible.
5 nine kinds of people liver CYP450 enzyme vitro inhibition effect rapid screening methods
Specific inhibitor proof test is carried out to above-mentioned set up mixed probe substrate system, by result being compared with single substrate system and existing Reported data, find all have good consistence, dependency is good, in table 4, show that built system is reliably feasible.The method that this mixed probe substrate is hatched altogether is utilized to replace the way of traditional Single probe substrate prediction CYP450 enzyme hypotype can well meet the early stage high-throughput requirement of medicament research and development, be applicable to screen the retarding effect of a large amount of drug candidate, consider the factors such as substrate selection, substrate interaction, solvent-susceptible degree, damping fluid and cofactor optimization simultaneously, make this mixed probe substrate Forecasting Methodology more reliable, for new drug development provides quick detection means easily.
Table 3. each meta-bolites mass spectrometric detection MRM parameter
Table 4 specific inhibitor suppresses IC50 value: single substrate, mixed probe substrate, literature values
Claims (3)
1. establish the rapid screening method of nine kinds of novel people liver CYP450 enzyme level effects, its feature comprises following several respects:
A) institute's establishment method can screen nine kinds of main CYP450 enzyme hypotypes, i.e. retarding effects of CYP1A2,2A6,2B6,2C8,2C9,2C19,2D6,2E1,3A4 simultaneously.
B) Specific probe selected by each hypotype is:
CYP1A2 Specific probe is Phenacetin (1.0 ~ 100 μMs);
CYP2A6 Specific probe is tonka bean camphor (0.1 ~ 2.3 μM);
CYP2B6 Specific probe is Bupropion (1.0 ~ 100 μMs);
CYP2C8 Specific probe is taxol (0.1 ~ 19 μM);
CYP2C9 Specific probe is tolbutamide (10 ~ 100 μMs), diclofenac (1.0 ~ 52 μMs);
CYP2C19 Specific probe is omeprazole (1.0 ~ 26 μMs), S-mephenytoin (0.1 ~ 35 μM);
CYP2D6 Specific probe is bufuralol (1 ~ 15 μM), Dextromethorphane Hbr (0.1 ~ 8.5 μM);
CYP2E1 Specific probe is chlorzoxazone (10 ~ 100 μMs);
CYP3A4 Specific probe is testosterone (10 ~ 94 μMs), midazolam (0.1 ~ 14 μM), Nifedipine (0.1 ~ 47 μM), omeprazole (1.0 ~ 26 μMs).
C) binding buffer liquid, organic solvent, BSA etc. on the impact of enzymic activity and substrate interaction etc. to selected each hypotype probe substrate do to divide into groups (as shown in the table) hatch, then detect multiple meta-bolites by LC-MS/MS simultaneously.
2. as claimed in claim 1, it is characterized in that considering damping fluid, organic solvent, BSA to factors such as the impact of enzymic activity and substrate interactions, incubated in vitro N-in-one technology is optimized.
3. used carrier behaviour hepatomicrosome in claim 1, is mainly used in metabolic enzyme retarding effect and differentiates.
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| CN105241992A (en) * | 2015-09-30 | 2016-01-13 | 成都华西海圻医药科技有限公司 | UPLC/MS/MS detection method of concentration of 4'-hydroxydiclofenac in liver microsome |
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| CN106770693A (en) * | 2015-11-19 | 2017-05-31 | 中国药科大学 | The external temperature of newtype drug is incubated, metabolin is found, drug-drug interactions prediction is quick screens and analytical integration strategy |
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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 |
| CN112098555A (en) * | 2020-09-16 | 2020-12-18 | 重庆市农业科学院 | Method for measuring enzymatic activities of CYP2A6, CYP2B6, CYP2C8 and CYP2D6 in earthworm |
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| CN112662733B (en) * | 2020-12-09 | 2023-08-29 | 安领生物医药(苏州)有限公司 | Screening method of metabolism new chemical entity liver CYP450 subtype enzyme |
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