CN106841421A - A kind of pharmacokinetics early stage druggability appraisal procedure - Google Patents
A kind of pharmacokinetics early stage druggability appraisal procedure Download PDFInfo
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Abstract
The invention discloses a kind of pharmacokinetics early stage druggability appraisal procedure, comprise the following steps：（1）MDR1 MDCKII cell screening models are set up, the transport features of medicine are studied；（2）The analysis method that UHPLC MS/MS determine rat plasma drug concentration is set up, the bioavilability of medicine is determined；（3）Set up inside and outside plasma protein binding rate assay method, the plasma protein binding characteristic of research medicine inside and outside（4）Medicine is investigated to six kinds of influences of rat liver microsomes cytochrome P 450 enzymes hypotype activity by " cocktail " hepatomicrosome incubated in vitro method；（5）By the hepatomicrosome method combination UHPLC MS/MS points of bioconversion situation of analysis of variance medicine.The method of the present invention can be used for the quick screening of new chemical entities pharmacokinetic properties, for early screening provides important decision information, improve the efficiency and quality of original new drug research and development.
Description
Technical field
The invention belongs to medicament research and development assessment technology field, in particular it relates to a kind of pharmacokinetics early stage druggability is commented
Estimate method.
Background technology
Original new drug research and development are the fields that a cycle is long, cost is big, risk is high, mortality is high.In recent years statistics shows,
During new drug development, the druggability mortality of new chemical entities (NCEs) is caused to be up to because pharmacokinetic property is undesirable
40%.Pharmacokinetics is related to the processes such as medicine absorption, distribution, metabolism and excretion in vivo, and new chemical entities medicine is for power
The early stage druggability assessment for learning characteristic has key effect.
The technology of drug absorption transport features research mainly includes in vitro method, in body method and intracorporal method, wherein in vitro method by
In large quantities of experimental animals are not needed, cost is few, and the test period is short, simple operation and other advantages and be widely used in noval chemical compound in early days
Screening.Caco-2 cell lines and MDR1-MDCKII cell lines in vitro method, can be spontaneously formed under Incubation Condition has
Cell polarity and tight internuncial cell monolayer, and the various carriers of secreting, expressing, are current widely used medicine bodies
Outer oral absorption screening model.
There are many drug absorption in-vitro screening models at present, but neither one external model can be simulated in organism completely
Complex environment, therefore, intracorporal method still have irreplaceable status, for the further research and development of noval chemical compound,
Carrying out the research of vivo biodistribution availability has important effect.
The plasma protein binding characteristic research of new drug has great importance to its further research and development and rationally application.The portion
Point the most frequently used research method is ultrafiltration and equilibrium dialysis, and relative to equilibrium dialysis, ultrafiltration has that equipment is simple, consumption
When short, plasma sample consumption it is few the advantages of.Cytochrome P450 mixed function oxidase system (cytochrome P450CYPs)
It is most important metabolic enzyme in liver, when a kind of medicine causes the induction of CYPs or suppresses, it is possible to which influence exists with medication thing
Disposal situation in organism, thus research influence of the medicine to drug metabolism enzymatic activity have important practical significance.
In the research and development of new medicinal products, medicine should be studied to the induction of metabolic enzyme particularly Cytochrome P450 (CYP450) enzyme or
Depression effect.Related external investigative technique mainly includes：Hepatomicrosome, liver S9, primary hepatocyte and P450 recombinases etc..It is biological
Study on Transformation is the indispensable important content of research and development of original new drug, can carry out pharmacological toxicology screening, and original new drug enters one
Step research provides important information.
At present, not seeing also has the simple and quick and comprehensive pharmacokinetics early stage druggability that carried out to new chemical entities to assess
Method.
The content of the invention
The technical problems to be solved by the invention are to overcome new drug pharmacokinetics early stage druggability in the prior art to assess
The defect and deficiency of technology, there is provided a kind of pharmacokinetics early stage druggability appraisal procedure, are conducive to new chemical entities medicine generation dynamic
The quick screening of mechanical characteristic, the transport features of preliminary examinations original new drug, absolute bioavailability, protein binding rate, to body
Influence, metabolite of outer CYP450 drug metabolisms enzymatic activity etc., important decision information is provided so as to be embodied as early screening
Target, for the research and development of its next step provide crucial foundation, can effectively improve the efficiency and quality of original new drug research and development.
It is an object of the invention to provide a kind of pharmacokinetics early stage druggability appraisal procedure.
Above-mentioned purpose of the invention is to give realization by the following technical programs.
A kind of pharmacokinetics early stage druggability appraisal procedure of medicine, comprises the following steps：
S1. the UHPLC-MS/MS analysis methods of medicine are first set up, MDR1-MDCKII cell screening models are resettled, is investigated
Transhipment of the P-gp inhibitor on medicine influences；
S2. set up and verify that UHPLC-MS/MS determines the analysis method of rat plasma drug concentration, and determine medicine and exist
Absolute bioavailability in rat body, calculates dynamic for kinetic parameter；
S3. medicine is separated using ultrafiltration, the inside and outside plasma protein binding rate of medicine is determined with reference to UHPLC-MS/MS；
S4. shadow of the medicine to rat liver microsomes cytochrome P 450 enzymes hypotype activity is determined by " cocktail " method
Ring；
S5. the bioconversion situation of medicine is analyzed using liver particle method and UHPLC-MS/MS.
Preferably, the pharmacokinetics early stage druggability appraisal procedure of said medicine, comprises the following steps：
S1. the UHPLC-MS/MS analysis methods of medicine to be assessed and positive drug are first set up, resettle and by positive drug come
Checking MDR1-MDCKII cell screening models, investigate transhipment influence of the P-gp inhibitor on medicine to be assessed；
S2. the UHPLC-MS/MS analysis methods for determining drug concentration to be assessed in rat plasma are set up and verify, by filling
Stomach or/and intravenously administrable determine absolute bioavailability of the medicine to be assessed in rat body, calculate dynamic with non-compartment model method
For kinetic parameter；
S3. medicine is separated using ultrafiltration, the inside and outside plasma protein knot of medicine to be assessed is determined with reference to UHPLC-MS/MS
Conjunction rate；
S4. the UHPLC-MS/MS analysis methods of external hepatomicrosome probe medicament are set up and is verified, is investigated with reference to positive drug
Medicine to be assessed is to six kinds of influences of rat liver microsomes cytochrome P 450 enzymes hypotype activity；
S5. by the biological specimen warp after external liver microsomes incubation sample and the rat intravenous injection administration of medicine to be assessed
UHPLC-MS/MS analyses are carried out after SPE, the structure and its metabolite of medicine to be assessed is determined, researched and analysed to be assessed
The bioconversion of medicine.
Wherein preferably, when medicine to be assessed is targeting anti-tumor Aurora A inhibitor Ly-7u or 8-hydroxyquinoline
During derivative 83b1, positive drug described in step S1 is digoxin.
Preferably, when medicine to be assessed is targeting anti-tumor Aurora A inhibitor Ly-7u or 8-hydroxyquinoline derives
During thing 83b1, P-gp inhibitor described in step S1 is Verapamil.
Preferably, when medicine to be assessed is targeting anti-tumor Aurora A inhibitor Ly-7u：Described in step S1 or S2
In UHPLC-MS/MS analysis methods,：Internal standard is Ly-7z, and Mass Spectrometry Conditions are：Ion gun ESI, Selective reaction monitoring pattern, prison
Measured ion is ESI+：M/z 410 → 353 (Ly-7u), m/z 457 → 368 (Ly-7z)；
Preferably, when medicine to be assessed is targeting anti-tumor Aurora A inhibitor Ly-7u：Described in step S1
In UHPLC-MS/MS analysis methods, liquid-phase condition is：Splitter XTerra MS C18 chromatographic columns；Mobile phase is：Volume ratio 1：9
0.1% formic acid water-methanol, flow velocity be 300 μ L/min, column temperature is room temperature, and analysis time is 3.0min；
Preferably, when medicine to be assessed is targeting anti-tumor Aurora A inhibitor Ly-7u：Described in step S2
In UHPLC-MS/MS analysis methods, liquid-phase condition is：Analytical column hypersil gold C18 chromatographic columns；Mobile phase is methyl alcohol
(A)-water (B), the ammonium acetate containing 0.1% formic acid and 5mM in water (B), A phases, B phase percentages sum are 100%；Eluent gradient
Elution requirement is：With the percentages of A phases, 0~1min, 5%~90%A；1~4min, 90%~90%A；4~4.1min,
90%~5%A；4.1~5min, 90%~90%A；Flow velocity is 300 μ L/min；Column temperature is room temperature, and analysis time is 5.0min.
Preferably, when medicine to be assessed is 8-hydroxyquinoline derivative 83b1, UHPLC-MS/MS described in step S1 or S2
Analysis method is：With Loratadine as internal standard, Mass Spectrometry Conditions are：Ion gun ESI, Selective reaction monitoring pattern, monitoring ion is
ESI+：m/z 321.96→m/z 162.84(83b1)；383.02 → m/z of m/z 259.12 (Loratadine)；Liquid-phase condition
For：Chromatographic column C18XTerra MS；Mobile phase：Volume ratio is 9：1 formic acid water of acetonitrile -1%；Flow velocity 0.3mLmin-1；Sample introduction
Measure 5 μ L；40 DEG C of column temperature, analysis time is 2.0min.
Preferably, when medicine to be assessed is 8-hydroxyquinoline derivative 83b1, positive drug described in step S1 is digoxin,
The condition of its UHPLC-MS/MS analysis method is as follows：
With foxalin (digitoxin) as internal standard；Mass Spectrometry Conditions are：Ion gun ESI, Selective reaction monitoring pattern
(SRM), monitoring ion is ESI-：M/z 825 → 779 (DG), m/z 809 → 763 (digitoxin)；Liquid-phase condition：
C18Hypersil gold MS analytical columns (5 μm, 2.1 × 100mm.Thermo, USA)；Mobile phase (contains for methanol-water
0.002% formic acid) (60/30, v/v)；300 μ L/min, column temperature is room temperature.Analysis time is 3.0min.
Preferably, when medicine to be assessed is targeting anti-tumor Aurora A inhibitor Ly-7u or 8-hydroxyquinoline derives
During thing 83b1, the condition of UHPLC-MS/MS analysis methods is described in step S4：Mass Spectrometry Conditions：Ion gun ESI, selection reaction prison
Survey pattern (SRM), monitoring ion is ESI+：M/z 152 → 110 (paracetamol), m/z 287 → 171 (4- hydroxy-methylbenzene sulphur fourths
Urea), m/z 362 → 214 (5- 5-Hydroxyomeprazoles)；M/z 258 → 157 (oxygen demethyl dextromethorphan), m/z 345 → 284
(oxidized nifedipine), m/z 383 → 262 (Loratadine)；ESI-：M/z 184 → 120 (6- hydroxyls Chlorzoxazone)；Liquid phase
Condition：Splitter C18XTerra MS analytical columns (5 μm, 100 × 2.1mm.Waters, USA)；Mobile phase is：Methyl alcohol：Water (contains
0.1% formic acid) (70：30, v/v), flow velocity is 300 μ L/min, and column temperature is room temperature, and analysis time is 3.5min.
Preferably, when medicine to be assessed is targeting anti-tumor Aurora A inhibitor Ly-7u or 8-hydroxyquinoline derives
During thing 83b1, positive drug described in step S4 is VX-680.
Preferably, when medicine to be assessed is targeting anti-tumor Aurora A inhibitor Ly-7u or 8-hydroxyquinoline derives
During thing 83b1, six kinds of rat liver microsomes cytochrome P 450 enzymes hypotypes described in step S4 be respectively CYP2E1, CYP2C9,
CYP1A2, CYP2C19, CYP2D1 and CYP3A4.
In addition, preferably can embodiment, the structure of MDR1-MDCKII cell screenings model described in step S1 as one kind
Method is：MDR1-MDCKII cells are inoculated on Transwell plates carries out monolayer cultivation 5 days, with digoxin as positive drug,
By investigate cross-film resistance, the apparent Penetration ration of fluorescein, digoxin two-way K+transport carry out cell monolayer model construction
And checking.
As it is a kind of preferably can embodiment, the specific method of step S2 is：SD rats 12 are grouped at random；Gavage is given
Medicine group：Gavage gives Ly-7u, and dosage is 25mg/kg；In (0h) before administration, 0.25 after administration, 0.5,1,2,3,4,5,8,12,
24th, 48,72h is from the μ L of right taking blood from jugular vein about 250；Intravenously administrable group：Single intravenous injection Ly-7u, dosage is 2.5mg/kg；In
0.05 after (0h) before administration, administration, 0.25,0.5,1,2,3,4,5,8,12,24,48h is from the μ L of right taking blood from jugular vein about 250；Profit
The concentration of medicine to be assessed in blood plasma is detected with UHPLC-MS/MS methods, main moving for dynamics is calculated with non-compartment model method
Parameter.
As it is a kind of preferably can embodiment, the specific method of step S3 is as follows：
(1) concentration of compound L y-7u is determined using UHPLC-MS/MS methods；Select low middle 3 concentration high (0.5,
5.0th, 50.0mg/L, n=3), Ly-7u is investigated with the external plasma protein binding rate of rat and people respectively；
(2) 12 SD rats (male and female half and half) be randomly divided into basic, normal, high three dosage groups (5mg/kg, 50mg/kg,
250mg/kg), tail vein injection gives the Ly-7u parenteral solutions of above-mentioned dosage.Certain hour collection 5mL venous blood, right after administration
Ly-7u plasma protein binding rates in rat body are investigated.
As it is a kind of preferably can embodiment, the specific method of step S4 is as follows：
(1) foundation of " cocktail " method：
Homogenate carries hepatomicrosome after taking out rats'liver, add medicine to be assessed, paracetamol, 4- hydroxy-methylbenzene sulphurs butyl urea,
5- 5-Hydroxyomeprazoles, oxygen demethyl dextromethorphan, oxidized nifedipine, internal standard and 6- hydroxyls Chlorzoxazone carry out incubated in vitro
Experiment.UHPLC-MS/MS detections paracetamol, 4- hydroxy-methylbenzene sulphurs butyl urea, 5- 5-Hydroxyomeprazoles, oxygen demethyl are right after incubation
The content of dextromethorphan, oxidized nifedipine, internal standard and 6- hydroxyl Chlorzoxazones this several drugses.The medicine of detection is metabolic enzyme
Substrate, after adding medicine to be assessed, if it has suppression to metabolic enzyme or induces, then with the medicament contg that this is metabolized through this enzyme
Will change, so as to judge influences of the 83b1 to enzyme.
Rat liver microsomes sample is extracted with 1mL ethyl acetate, and Loratadine is internal standard, using UHPLC-MS/MS methods
It is analyzed；The condition of UHPLC-MS/MS is as follows：
Mass Spectrometry Conditions are：Ion gun ESI, Selective reaction monitoring pattern (SRM), monitoring ion is ESI+：m/z 152→
110 (paracetamols), m/z 287 → 171 (4- hydroxy-methylbenzene sulphurs butyl urea), m/z 362 → 214 (5- 5-Hydroxyomeprazoles)；m/z
258 → 157 (oxygen demethyl dextromethorphans), m/z 345 → 284 (oxidized nifedipine), m/z 383 → 262 (internal standard chlorine thunder he
It is fixed)；ESI-：M/z 184 → 120 (6- hydroxyls Chlorzoxazone)；
Liquid-phase condition：Splitter C18XTerra MS analytical columns (5 μm, 100 × 2.1mm.Waters, USA)；Mobile phase
For：Volume ratio is 7：3 methyl alcohol：The aqueous solution, contains 0.1% formic acid in the aqueous solution, flow velocity is 300 μ L/min, and column temperature is room temperature, point
The analysis time is 3.5min；
The medicine of detection is the substrate of metabolic enzyme, after adding 83b1, if it has suppression to metabolic enzyme or induces, then with
This will change through the medicament contg that this enzyme is metabolized；
(2) " two step method " screening strategy is used, medicine to be assessed and positive drug is investigated to six kinds of CYP450 enzyme hypotypes
The In-vitro Inhibitory Effect of CYP2E1, CYP2C9, CYP1A2, CYP2C19, CYP2D1, CYP3A4 and corresponding IC50Value.
Preferably, the measure of the structure of medicine to be assessed described in step S5 and its metabolite uses one-level full scan matter
Spectrum, Selective reaction monitoring and second order mses scan mode are detected simultaneously.
In addition, application of the above method in the quick screening of new chemical entities pharmacokinetic properties is also protected in the present invention
In the range of shield.
The present invention compared with prior art, has the advantages that：
(1) the MDR1-MDCKII cell models that the present invention sets up can be quickly commented the transport features of new chemical entities
Valency；
(2) intracorporal method that the present invention is used carries out new drug early stage bioavailability study, simple to operate, efficient.
(3) ultrafiltration of the present invention carries out inside and outside plasma protein binding rate research, and feasibility is strong, and stability is high；
(4) multiprobe substrate method of the present invention, i.e. " cocktail " method investigate medicine to rat liver microsomes body cell
The In-vitro Inhibitory Effect of cytochrome p 450 enzyme, it is efficiently quick；
(5) present invention uses high-resolution UHPLC-QE-MS/MS, and high resolution is more suitable for metabolite identification.
Specific embodiment
The present invention is made with reference to specific embodiment further being elaborated, the embodiment is served only for explaining this
Invention, is not intended to limit the scope of the present invention.Test method used in following embodiments unless otherwise specified, is often
Rule method；Material, reagent for being used etc., are the reagent and material for commercially obtaining unless otherwise specified.
The early stage pharmacokinetics druggability assessment of the novel targeted antitumor Aurora A inhibitor Ly-7u of embodiment 1
Compound L y-7u is the achievement in research of the present inventor team early stage, and its structural formula is as follows：
In vitro study shows that the compound has good antitumor activity, and is lived with Aurora A/B kinase inhibitions
Property and selectivity, with good application prospect.The assessment of early stage pharmacokinetics druggability is carried out below for the compound.
1st, MDR1-MDCKII cell models are built to study the transport features of Ly-7u
(1) foundation of the UHPLC-MS/MS analysis methods of testing compound Ly-7u：
With Ly-7z as internal standard, its structural formula is as follows：
Mass Spectrometry Conditions are：Ion gun ESI, Selective reaction monitoring pattern (SRM), monitoring ion is ESI+：m/z 410→
353 (Ly-7u), m/z 457 → 368 (Ly-7z).
Liquid-phase condition：Splitter XTerra MS C18 chromatographic columns (5 μm, 2.1 × 100mm.Waters, USA)；Mobile phase
For：0.1% formic acid water-methanol (1/9, v/v), flow velocity is 300 μ L/min, and column temperature is room temperature.Analysis time is 3.0min.
(2) foundation of positive control drug digoxin (DG) UHPLC-MS/MS analysis methods：
With foxalin (digitoxin) as internal standard.
Mass Spectrometry Conditions are：Ion gun ESI, Selective reaction monitoring pattern (SRM), monitoring ion is ESI-：m/z 825→
779 (DG), m/z 809 → 763 (digitoxin).
Liquid-phase condition：C18Hypersil gold MS analytical columns (5 μm, 2.1 × 100mm.Thermo, USA)；Mobile phase
It is methanol-water (containing 0.002% formic acid) (60/30, v/v)；300 μ L/min, column temperature is room temperature.Analysis time is 3.0min.
(3) foundation of MDR1-MDCKII cell screenings model：
MDR1-MDCKII cells are inoculated on Transwell plates and carry out monolayer cultivation 5 days, with digoxin as positive drug, lead to
Cross investigation cross-film resistance, the apparent Penetration ration of fluorescein, the two-way K+transport of digoxin carry out cell monolayer model construction and
Checking, successfully builds MDR1-MDCKII cell models.
(4) application of MDR1-MDCKII cell models：
With the two-way transhipment of MDR1-MDCKII cell screening scale-model investigations Ly-7u, P-gp inhibitor Verapamils pair are investigated
The influence of its two-way transhipment.
The transport experiment result of Ly-7u shows, not plus during P-gp inhibitor Verapamils, it is big that the outer row of Ly-7u leads ER
In 3, outer row leads ER and is remarkably decreased after addition inhibitor Verapamil, points out the substrate that Ly-7u is efflux protein P-gp；Add
After Verapamil, row leads ER less than 1 outside Ly-7u, illustrates that the transporting mode of Ly-7u is mainly active transport.
2nd, oral absolute bioavailability researchs of the Ly-7u in rat body
(1) foundation of the UHPLC-MS/MS analysis methods of compound L y-7u：
With Ly-7z as internal standard.
Mass Spectrometry Conditions are：Ion gun ESI, Selective reaction monitoring pattern (SRM), monitoring ion is ESI+：m/z 410→
353 (Ly-7u), m/z 457 → 368 (Ly-7z).
Liquid-phase condition：Analytical column hypersil gold C18 chromatographic columns (2.1mm × 100mm, 1.9 μm；Thremo,
USA)；
Mobile phase is：Methyl alcohol (A)-water (B), the ammonium acetate containing 0.1% formic acid and 5mM, A phases, B phase percentages in water (B)
Sum is 100%；
Eluent gradient elution requirement：0~1min, 5%~90%A (with the percentages of A phases)；1~4min, 90%~
90%A；4~4.1min, 90%~5%A；4.1~5min, 90%~90%A；
Flow velocity is 300 μ L/min；Column temperature is room temperature.Analysis time is 5.0min.
(2) absolute bioavailability researchs of the Ly-7u in rat body：
SD rats 12 are grouped at random.
Gastric infusion group：Gavage gives Ly-7u, and dosage is 25mg/kg；In (0h) before administration, 0.25 after administration, 0.5,1,
2nd, 3,4,5,8,12,24,48,72h is from the μ L of right taking blood from jugular vein about 250.
Intravenously administrable group：Single intravenous injection Ly-7u, dosage is 2.5mg/kg；In (0h) before administration, 0.05 after administration,
0.25th, 0.5,1,2,3,4,5,8,12,24,48h is from the μ L of right taking blood from jugular vein about 250.
Ly-7u concentration in UHPLC-MS/MS methods detection blood plasma, calculates main moving and joins for dynamics with non-compartment model method
Number.
(3) according to rat single oral and the resulting estimate of intravenous injection, after the administration of SD rats single oral gavage (25mg/kg)
The absolute bioavailability of Ly-7u is about 28.7% ± 9.7%.
3rd, the research of Ly-7u inside and outsides plasma protein binding rate is carried out using ultrafiltration
(1) medicine is separated using ultrafiltration；The concentration of compound L y-7u is determined using UHPLC-MS/MS methods；Selection is low
Middle 3 concentration high (0.5,5.0,50.0mg/L, n=3), respectively to Ly-7u and the external plasma protein binding rate of rat and people
Investigated.
(2) 12 SD rats (male and female half and half) be randomly divided into basic, normal, high three dosage groups (5mg/kg, 50mg/kg,
250mg/kg), tail vein injection gives the Ly-7u parenteral solutions of above-mentioned dosage.Certain hour collection 5mL venous blood, right after administration
Ly-7u plasma protein binding rates in rat body are investigated.
(3) result shows that low, middle dose group SD rats Ly-7u internal plasma protein binding rate does not have obvious dosage
Correlation, and high dose group is substantially lower than low, middle dose group.Tail vein injection gives basic, normal, high dosage to SD rats respectively
After (respectively 5mg/kg, 50mg/kg, 250mg/kg dosage group), Ly-7u plasma protein binding rates in vivo are respectively
50.7% ± 1.9%, 49.7% ± 3.1%, 46.4% ± 3.9%.
4th, influences of " cocktail " the method research Ly-7u to external CYP450 drug metabolisms enzymatic activity
(1) foundation of " cocktail " method：
Homogenate carries hepatomicrosome after taking out rats'liver, adds Ly-7u, paracetamol, 4- hydroxy-methylbenzene sulphurs butyl urea, 5- hydroxyls
Omeprazole, oxygen demethyl dextromethorphan, oxidized nifedipine, internal standard and 6- hydroxyls Chlorzoxazone carry out incubated in vitro experiment.
UHPLC-MS/MS detections paracetamol, 4- hydroxy-methylbenzene sulphurs butyl urea, 5- 5-Hydroxyomeprazoles, oxygen demethyl are right U.S. husky after incubation
The content of sweet smell, oxidized nifedipine, internal standard and 6- hydroxyl Chlorzoxazones this several drugses.The medicine of detection is the bottom of metabolic enzyme
Thing, after adding 83b1, if it has suppression to metabolic enzyme or induces, then will be become through the medicament contg that this enzyme is metabolized with this
Change, so as to judge influences of the 83b1 to enzyme.
Rat liver microsomes sample is extracted with ethyl acetate (1mL), and Loratadine is internal standard, using UHPLC-MS/MS side
Method is analyzed；The condition of UHPLC-MS/MS is as follows：
Mass Spectrometry Conditions are：Ion gun ESI, Selective reaction monitoring pattern (SRM), monitoring ion is ESI+：m/z 152→
110 (paracetamols), m/z 287 → 171 (4- hydroxy-methylbenzene sulphurs butyl urea), m/z 362 → 214 (5- 5-Hydroxyomeprazoles).m/z
258 → 157 (oxygen demethyl dextromethorphans), m/z 345 → 284 (oxidized nifedipine), m/z 383 → 262 (internal standard)；
ESI-：M/z 184 → 120 (6- hydroxyls Chlorzoxazone).
Liquid-phase condition：Splitter C18XTerra MS analytical columns (5 μm, 100 × 2.1mm.Waters, USA)；Mobile phase
For：Methyl alcohol：Water (containing 0.1% formic acid) (70：30, v/v), flow velocity is 300 μ L/min, and column temperature is room temperature.Analysis time is
3.5min。
(2) " two step method " screening strategy is used, compound L y-7u and positive drug VX-680 is investigated sub- to six kinds of CYP450 enzymes
Type CYP2E1, CYP2C9, the In-vitro Inhibitory Effect of CYP1A2, CYP2C19, CYP2D1, CYP3A4 and corresponding IC50Value.
(3) result shows：Ly-7u is to six kinds of equal unrestraint effects of enzyme hypotype；Positive drug VX-680 is CYP1A2 (IC50
=25.6 μM), 2C9 (IC50=30.3 μM), 2D6 (IC50=36.5 μM), the weak suppression of 2E1 (IC50=34.1 μM) enzyme hypotype
Agent.
5th, in body and the bioconversion of external hepatomicrosome method research Ly-7u
(1) biological specimen after the external liver microsomes incubation samples of compound L y-7u and rat intravenous injection are administered, through solid
UHPLC-MS/MS analyses are carried out after mutually extracting.
Analysis to Ly-7u structures, it is same using one-level full scan mass spectrum, Selective reaction monitoring and second order mses scan mode
Shi Jinhang is detected.
(2) result shows：Searching out four kinds of Ly-7u may metabolite.
The early stage pharmacokinetics druggability assessment of the 8-hydroxyquinoline derivative (83b1) of embodiment 2
83b1 is a kind of 8-hydroxyquinoline derivative, is a kind of potential new drug that inventor team early-stage Study goes out, chemical combination
The structural formula of thing is as follows：
The assessment of early stage pharmacokinetics druggability is carried out below for the compound.
1st, MDR1-MDCKII cell models are studied the transport features of 83b1
(1) foundation of compound 83b1UHPLC-MS/MS analysis methods：
With Loratadine as internal standard.
Mass Spectrometry Conditions are：Ion gun ESI, Selective reaction monitoring pattern (SRM), monitoring ion is ESI+：m/z 321.96
→m/z 162.84(83b1)；383.02 → m/z of m/z 259.12 (Loratadine).
Liquid-phase condition：Chromatographic column C18XTerra MS (5 μm, 150 × 2.1mm.Waters, USA)；Mobile phase：Acetonitrile-
1% formic acid water (9:1, v/v)；Flow velocity 0.3mLmin-1；The μ L of sample size 5；40 DEG C of column temperature, analysis time is 2.0min.
(2) digoxin (DG) UHPLC-MS/MS analysis methods set up same as Example 1.
(3) foundation of MDR1-MDCKII cell screenings model：It is identical with embodiment 1.
(4) application of MDR1-MDCKII cell screenings model：
With the two-way transhipment of MDR1-MDCKII cell screening scale-model investigations 83b1, P-gp inhibitor Verapamils pair are investigated
The influence of its two-way transhipment.
The transport experiment result of 83b1 shows that, when P-gp inhibitor Verapamils are not added, the outer row of 83b1 leads ER and is more than
4, outer row leads ER and is remarkably decreased after addition inhibitor Verapamil, points out the substrate that 83b1 is efflux protein P-gp.
2nd, oral absolute bioavailability researchs of the 83b1 in rat body
(1) foundation of the UHPLC-MS/MS analysis methods of compound 83b1 is identical with step 1.
(2) absolute bioavailability researchs of the 83b1 in rat body：
SD rats 12 are grouped at random.
Gastric infusion group：Gavage gives 83b1, and dosage is 10mg/kg；In (0h) before administration, 0.25 after administration, 0.5,1,
2nd, 3,4,5,8,12,24,48,72h is from the μ L of right taking blood from jugular vein about 300.
Intravenously administrable group：Single intravenous injection 83b1, dosage is 10mg/kg；In (0h) before administration, 0.05 after administration,
0.25th, 0.5,1,2,3,4,5,8,12,24,48h is from the μ L of right taking blood from jugular vein about 300.
83b1 concentration in UHPLC-MS/MS methods detection blood plasma, calculates main moving and joins for dynamics with non-compartment model method
Number.
(3) according to rat single oral and the resulting estimate of intravenous injection, after the administration of SD rats single oral gavage (25mg/kg)
The absolute bioavailability of 83b1 is about 20.9% ± 8.8%.
3rd, the research of 83b1 inside and outsides plasma protein binding rate is carried out using ultrafiltration
(1) medicine is separated using ultrafiltration；The concentration of compound 83b1 is determined using UHPLC-MS/MS methods；Selection is low
Middle 3 concentration high (0.1,10.0,100.0mg/L, n=3), respectively to 83b1 and the external plasma protein binding rate of rat and people
Investigated.
(2) 12 SD rats (male and female half and half) be randomly divided into basic, normal, high three dosage groups (1mg/kg, 10mg/kg,
100mg/kg), tail vein injection gives the 83b1 parenteral solutions of above-mentioned dosage.Certain hour collection 5mL venous blood, right after administration
83b1 plasma protein binding rates in rat body are investigated.
(3) result shows, tail vein injection gives basic, normal, high dosage (respectively 1mg/kg, 10mg/ to SD rats respectively
Kg, 100mg/kg dosage group) after, 83b1 plasma protein binding rates in vivo are respectively 32.7% ± 2.2%, 31.7% ±
3.2%th, 26.7% ± 4.3%.
4th, influences of " cocktail " the method research 83b1 to external CYP450 drug metabolisms enzymatic activity
(1) foundation of " cocktail " method：
Homogenate carries hepatomicrosome after taking out rats'liver, adds 83b1, paracetamol, 4- hydroxy-methylbenzene sulphurs butyl urea, 5- hydroxyls
Omeprazole, oxygen demethyl dextromethorphan, oxidized nifedipine, internal standard and 6- hydroxyls Chlorzoxazone carry out incubated in vitro experiment.
UHPLC-MS/MS detections paracetamol, 4- hydroxy-methylbenzene sulphurs butyl urea, 5- 5-Hydroxyomeprazoles, oxygen demethyl are right U.S. husky after incubation
The content of sweet smell, oxidized nifedipine, internal standard and 6- hydroxyl Chlorzoxazones this several drugses.The medicine of detection is the bottom of metabolic enzyme
Thing, after adding 83b1, if it has suppression to metabolic enzyme or induces, then will be become through the medicament contg that this enzyme is metabolized with this
Change, so as to judge influences of the 83b1 to enzyme.
Rat liver microsomes sample is extracted with ethyl acetate (1mL), and Loratadine is internal standard, using UHPLC-MS/MS side
Method is analyzed；The condition of UHPLC-MS/MS is as follows：
Mass Spectrometry Conditions are：Ion gun ESI, Selective reaction monitoring pattern (SRM), monitoring ion is ESI+：m/z 152→
110 (paracetamols), m/z 287 → 171 (4- hydroxy-methylbenzene sulphurs butyl urea), m/z 362 → 214 (5- 5-Hydroxyomeprazoles).m/z
258 → 157 (oxygen demethyl dextromethorphans), m/z 345 → 284 (oxidized nifedipine), m/z 383 → 262 (internal standard)；
ESI-：M/z 184 → 120 (6- hydroxyls Chlorzoxazone).Liquid-phase condition：Splitter C18XTerra MS analytical columns (5 μm, 100 ×
2.1mm.Waters,USA)；Mobile phase is：Methyl alcohol：Water (containing 0.1% formic acid) (70：30, v/v), flow velocity is 300 μ L/min, post
Temperature is room temperature.Analysis time is 3.5min.
(2) " two step method " screening strategy is used, compound 83b1 and positive drug VX-680 is investigated sub- to six kinds of CYP450 enzymes
Type CYP2E1, CYP2C9, the In-vitro Inhibitory Effect of CYP1A2, CYP2C19, CYP2D1, CYP3A4 and corresponding IC50Value.
(3) result shows：83b1 is to the equal unrestraint effect of five kinds of enzyme hypotypes in addition to CYP2C9 (IC50=37.8 μM)；
83b1 is the weak inhibitor of CYP2C9 (IC50=37.8 μM) enzyme hypotype.
5th, in body and the bioconversion of external hepatomicrosome method research 83b1
(1) biological specimen after the external liver microsomes incubation samples of compound 83b1 and rat intravenous injection are administered, through solid
UHPLC-MS/MS analyses are carried out after mutually extracting.
Analysis to 83b1 structures, it is same using one-level full scan mass spectrum, Selective reaction monitoring and second order mses scan mode
Shi Jinhang is detected.
(2) result shows：Searching out seven kinds of 83b1 may metabolite.
Protection of the invention is not limited to above example.
Claims (10)
1. the pharmacokinetics early stage druggability appraisal procedure of a kind of medicine, it is characterised in that comprise the following steps：
S1. the UHPLC-MS/MS analysis methods of medicine to be assessed are first set up, MDR1-MDCKII cell screening models are resettled, is examined
Examine transhipment influence of the P-gp inhibitor on medicine to be assessed；
S2. set up and verify that UHPLC-MS/MS determines the analysis method of rat plasma drug concentration, and determine medicine in rat
Internal absolute bioavailability, calculates pharmacokinetic parameter；
S3. medicine is separated using ultrafiltration, the inside and outside plasma protein binding rate of medicine is determined with reference to UHPLC-MS/MS；
S4. influence of the medicine to rat liver microsomes cytochrome P 450 enzymes hypotype activity is determined by " cocktail " method；
S5. the bioconversion situation of medicine is analyzed using liver particle method and UHPLC-MS/MS.
2. the pharmacokinetics early stage druggability appraisal procedure of medicine according to claim 1, it is characterised in that including as follows
Step：
S1. the UHPLC-MS/MS analysis methods of medicine to be assessed and positive drug are first set up, is resettled and is verified by positive drug
MDR1-MDCKII cell screening models, investigate transhipment influence of the P-gp inhibitor on medicine to be assessed；
S2. the UHPLC-MS/MS analysis methods for determining drug concentration to be assessed in rat plasma are set up and verify, by gavage
Or/and intravenously administrable determines absolute bioavailability of the medicine to be assessed in rat body, dynamic generation is calculated with non-compartment model method
Kinetic parameter；
S3. medicine is separated using ultrafiltration, the inside and outside plasma protein for determining medicine to be assessed with reference to UHPLC-MS/MS is combined
Rate；
S4. the UHPLC-MS/MS analysis methods of external hepatomicrosome probe medicament are set up and is verified, investigates to be evaluated with reference to positive drug
Medicine is estimated to six kinds of influences of rat liver microsomes cytochrome P 450 enzymes hypotype activity；
S5. by the biological specimen after external liver microsomes incubation sample and the rat intravenous injection administration of medicine to be assessed through solid phase
UHPLC-MS/MS analyses are carried out after extraction, the structure and its metabolite of medicine to be assessed is determined, medicine to be assessed is researched and analysed
Bioconversion.
3. method according to claim 2, it is characterised in that positive drug described in step S1 is digoxin.
4. method according to claim 2, it is characterised in that P-gp inhibitor described in step S1 is Verapamil.
5. method according to claim 2, it is characterised in that when medicine to be assessed is the suppression of targeting anti-tumor Aurora A
During preparation Ly-7u：
In UHPLC-MS/MS analysis methods described in step S1 or S2,：Internal standard is Ly-7z, and Mass Spectrometry Conditions are：Ion gun ESI,
Selective reaction monitoring pattern, monitoring ion is ESI+：M/z 410 → 353 (Ly-7u), m/z 457 → 368 (Ly-7z)；
In UHPLC-MS/MS analysis methods described in step S1, liquid-phase condition is：Splitter XTerra MS C18 chromatographic columns；Flowing
Xiang Wei：Volume ratio 1：9 0.1% formic acid water-methanol, flow velocity is 300 μ L/min, and column temperature is room temperature, and analysis time is 3.0min；
In UHPLC-MS/MS analysis methods described in step S2, liquid-phase condition is：Analytical column hypersil gold C18 chromatograms
Post；Mobile phase is methyl alcohol (A)-water (B), the ammonium acetate containing 0.1% formic acid and 5mM in water (B), and A phases, B phase percentage sums are
100%；Eluent gradient elution requirement is：With the percentages of A phases, 0~1 min, 5%~90% A；1~4 min, 90%~
90% A；4~4.1 min, 90%~5% A；4.1~5 min, 90%~90% A；Flow velocity is 300 μ L/min；Column temperature is room
Temperature, analysis time is 5.0 min.
6. method according to claim 2, it is characterised in that when medicine to be assessed is 8-hydroxyquinoline derivative 83b1
When, UHPLC-MS/MS analysis methods described in step S1 or S2 are：With Loratadine as internal standard, Mass Spectrometry Conditions are：Ion gun ESI,
Selective reaction monitoring pattern, monitoring ion is ESI+：m/z 321.96→m/z 162.84(83b1)；m/z 383.02→m/z
259.12 (Loratadines)；Liquid-phase condition is：Chromatographic column C18 XTerra MS；Mobile phase：Volume ratio is 9：1 first of acetonitrile -1%
Sour water；The mLmin of flow velocity 0.3-1；The μ L of sample size 5；40 DEG C of column temperature, analysis time is 2.0 min.
7. method according to claim 2, it is characterised in that when medicine to be assessed is targeting anti-tumor Aurora A
During inhibitor Ly-7u or 8-hydroxyquinoline derivative 83b1, the condition of UHPLC-MS/MS analysis methods is described in step S4：Matter
Spectral condition：Ion gun ESI, Selective reaction monitoring pattern（SRM）, monitoring ion is ESI+：m/z 152→110（Paracetamol）,
M/z 287 → 171 (4- hydroxy-methylbenzene sulphurs butyl urea), m/z 362 → 214（5- 5-Hydroxyomeprazoles）；m/z 258→157（Oxygen goes
Methyl dextromethorphan）, m/z 345 → 284（Oxidized nifedipine）, m/z 383 → 262（Loratadine）；ESI-：m/z 184
→120（6- hydroxyl Chlorzoxazones）；Liquid-phase condition：Splitter C18 XTerra MS analytical columns（5 μm, 100×2.1mm.
Waters, USA）；Mobile phase is：Methyl alcohol：Water（Containing 0.1% formic acid）（70：30, v/v）, flow velocity is 300 μ L/min, and column temperature is room
Temperature, analysis time is 3.5 min.
8. method according to claim 2, it is characterised in that positive drug described in step S4 is VX-680.
9. method according to claim 2, it is characterised in that six kinds of rat liver microsomes cytochromes described in step S4
P450 enzyme hypotypes are respectively CYP2E1, CYP2C9, CYP1A2, CYP2C19, CYP2D1 and CYP3A4.
10. method according to claim 2, it is characterised in that outer hepatomicrosome probe medicament described in step S4 is to flutter heat
Breath pain, 4- hydroxy-methylbenzene sulphurs butyl urea, 5- 5-Hydroxyomeprazoles, oxygen demethyl dextromethorphan, oxidized nifedipine, Loratadine and
6- hydroxyl Chlorzoxazones.
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