CN103937869A - Kit for screening CYP450 enzyme inhibition medicament with high flux and research method thereof - Google Patents

Abstract

The invention relates to a kit for screening a CYP450 enzyme inhibition medicament with high flux and a research method thereof, and belongs to the field of medicament safety evaluation. A kit body of the kit comprises an NADPH regeneration system, a 0.1M phosphate buffer solution, six mixed probe substrates, metabolites of the six mixed probe substrates, and 20mg/ml of liver microsomes of a mouse, rat, dog or monkey. The NADPH regeneration system, six enzyme substrate and medicaments with different concentrations are mixed, then the mixture and the liver microsomes of the mouse, rat, dog or monkey are incubated in a water bath at a temperature of 37 DEG C together, the inhibition effects of the medicaments on the activity of a CYP450 enzyme are judged by LC-MS/MS detection of the quantities of the six metabolites, and the IC50 value is calculated. The kit has the advantages of simplicity, accuracy, stability and convenience, so that the kit has broad application prospect.

Description

A kind of test kit and research method thereof of high flux screening CYP450 enzyme inhibitor thing

Technical field

The test kit and the research method thereof that the present invention relates to a kind of high flux screening CYP450 enzyme inhibitor thing, belong to drug safety evaluation field.

Background technology

Cytochrome P450 (CYP450) belongs to a kind of oxyphorase fermentoid, is extensively present in organism, is most important gang oxydase in microsomal mixed function oxidase system, be distributed in Various Tissues, organ, relative molecular mass 50kDa, has maximum absorption band at wavelength 450nm place.The biochemical reaction that its participates in has the bio-oxidation etc. of oxidative metabolism, xenobiotics and most of medicine of the Endogenous Substrates such as lipid acid, steroid biosynthesizing and steroid hormone; It makes the xenobiotics that enters body develop through the backward both direction of metabolism: metabolism activation and metabolic detoxification, the product after metabolism activation has stronger toxicity, even produces teratogenesis, carcinogenic effect.

Cytochrome P450 is as the important enzyme relevant with drug metabolism, in drug metabolism, play considerable effect, the research of its metabolism will be contributed to the exploitation of new drug, and can help people to be more clearly familiar with the pathways metabolism of medicine, reduce the side effect of new drug in human body, increase curative effect of medication.The verified main causes of death that cause of medicine mechanism are exactly bad drug interaction, and when two or more Drug combination, the effect or the toxicity that change one of them medicine cause occurring bad drug interaction.A research of the U.S. shows, the serious adverse reaction incidence of inpatient is 6.7%, because of the lethality rate of the drug interaction 4-5 position of the rank inpatient cause of death, in nearly 20 years, U.S. FDA is successively withdrawn from 13 kinds of new drugs of approved listing from market, its topmost reason is exactly to have occurred serious Metabolic Drug Interactions.So, if a medicine produces and suppresses Cytochrome P450, in the process of drug combination, will cause Cytochrome P450 to be obstructed to the metabolism of another medicine, thereby produce toxicity, harm health, one of interaction of serious drug metabolism that Here it is, thereby in early stage R&D process, whether research compound has inhibition quite important to Cytochrome P450, for the clinical application of medicine, there is important meaning.

Due to the appearance of combinatorial chemistry and and fast-developing, making syntheticly has the compound speed of potential clinical treatment function also greatly to improve in a large number.Yet assess these compounds and lack corresponding quick detection kit and technical support, scientific research personnel need to be made by oneself or autogamy related reagent and microsome, the research method that enzyme suppresses is disunity and standard also, have single enzyme, also have a plurality of enzymes, all these are for the medical worker who does not pass through drug metabolism study training, carrying out related drugs interaction appraisal meeting is difficult to, even if done, also often waste a large amount of manpowers, material resources and after the time, but can prepare incorrect because of reagent, operate lack of standardization, calculate the information that the reason such as unprofessional causes obtaining drug interaction accurately, thereby underestimate the interaction risk of medicine when clinical application, potential hazard is larger.And enzymic activity detection kit both domestic and external is mainly the detection on single enzymic activity impact, and conventionally use fluorescent quantitation, HPLC detects or ELISA detection kit, detection sensitivity is low, error is larger, complicated operation, and far can not meet the requirement of high-flux fast screening, for evaluating, the interactional risk data of clinical drug is not comprehensive, can not simulate the real human body medication situation of integral body afterwards, therefore this area need to have test kit and the technological method of a set of thoroughly evaluating drug interaction especially, the risk of simple evaluation drug interaction fast, can meet the needs of the drug evaluation of the researcher that there is no associated metabolic profession basis especially, according to test kit specification sheets, operation can complete evaluation task.

Summary of the invention

The test kit and the research method thereof that the object of this invention is to provide a kind of high flux screening CYP450 enzyme inhibitor thing, test kit provides packing box and complete reagent collocation, can utilize the working instructions of test kit to carry out fast and accurately drug interaction research, utilize high-throughout LC-MS/MS detection technique to detect the amount of each meta-bolites, calculate IC ₅₀value, evaluates medicine to each CYP450 enzyme inhibition.This detection kit and research method thereof are mainly to provide technical support for medicine early screening and clinical application repercussion study, in particular for those, rigidly connect the researcher that touches drug metabolism study, clinical medical personnel provides material and the technical support of drug evaluation, can greatly shorten search time, increase work efficiency, promote China in the R&D work in clinical application field, for the quality of life that improves people, promote medical model, therapeutic modality and medication structural reform and play huge pushing effect.

In order to solve the existing problem of available reagent box and technical study, the present invention is by the following technical solutions: according to test kit specification sheets preparation NADPH regeneration system rapidly, after different concns medicine, mixed substrates and hepatomicrosome are hatched jointly, utilize LC-MS/MS to detect six meta-bolitess that FDA recommends, IC simultaneously ₅₀the calculating of value is carried out according to GraphPad Prism 5 Project softwares.IC ₅₀>50 μ mol/L explanation medicine is to CYP450 enzyme unrestraint effect, without the risk of drug interaction, 10<IC ₅₀<50 μ mol/L explanation medicine is weak inhibitor, and the risk of drug interaction is less, 1<IC ₅₀<10 μ mol/L explanation medicine is medium inhibitor, IC ₅₀<1 μ mol/L explanation medicine is potent inhibitor, and medium and potent inhibitor has the risk of drug interaction, needs further research and pays close attention to..

Technical scheme is specifically implemented:

(a) test kit of the present invention has NADPH regeneration system rapidly: NADPH regeneration system rapidly comprises A liquid: 2.5mmol/L NADPNa2,10U/mL6-glucose phosphate dehydrogenase, 20mmol/L magnesium chloride, B liquid: 50mmol/L6-glucose 1-phosphate1-.0.1mol/L phosphoric acid buffer comprises 29g Sodium phosphate dibasic, 2.96g SODIUM PHOSPHATE, MONOBASIC, and 42.5g sodium-chlor, 11.8g Repone K is dissolved in 1 liter of distilled water.Then by drawing 50 μ LB liquid+180, μ LA liquid+10 μ L0.1mol/L phosphoric acid buffers as NADPH regeneration system rapidly.

(b) test kit of the present invention has mixed substrates and hepatomicrosome: mixed probe substrate is Phenacetin (50 μ mol/L) at 250 μ L incubation system final concentrations, midazolam (2.5 μ mol/L), tolbutamide (120 μ mol/L), Dextromethorphane Hbr (5 μ mol/L), Bupropion (25 μ mol/L), mephenytoin (50 μ mol/L).The hepatomicrosome of mouse, rat, dog or monkey final concentration of protein in 250 μ L incubation systems is 0.4mg/ml.Test kit specification sheets also provides the suggestion final concentration of medicine series at 250 μ L incubation system reasonable preparations: 0.05,0.15,0.5,1.5,5,15,50,100 μ mol/L, hepatomicrosome and the NADPH regeneration system rapidly of final 2.5 μ L medicine series concentration+5, μ L mixed probe substrate+2.5 μ L20mg/ml mouse, rat, dog or monkey are hatched 1h in 37 ℃ of water-baths altogether.

(c) test kit specification sheets provides foundation can detect the LC-MS/MS method of six meta-bolitess simultaneously, by detecting the amount of six meta-bolitess, evaluate the restraining effect of medicine to CYP450 enzymic activity: hatch after 1h, with 3 times of (methyl alcohol: acetonitrile=1:1) protein precipitation, vortex concussion 2 minutes, 14000 leave the heart 10 minutes, get supernatant sample introduction in LC-MS/MS, detect the amount of six kinds of enzymes metabolism products.Liquid-phase condition: with mobile phase A (containing the pure water of 0.1% formic acid and 5mM ammonium formiate) and B(acetonitrile) gradient elution: 30%B(0min), 85%B(1.5-4.5min), 30%B(4.6min); 25 ℃ of column temperatures, flow velocity is 0.35mLmin-1, working time 5min; Sampling volume 10 μ L.Mass spectrum condition: in ESI source positive ion MRM mode, detect, 320 ℃ of capillary temperatures, capillary voltage+4000V, atomization voltage 25psi, dry gas flow velocity 10L/min, other mass spectroscopy parameter is in Table 1.

The ion pair of table 1 meta-bolites

Compound

Paracetamol

4-hydroxytoluene sulphur butyl urea

4-hydroxyl mephenytoin

Levorphanol d-form

Hydroxyl Bupropion

1'-hydroxyl midazolam

Ion pair

152.1/110.1

287/171.1

235.2/150.1

258.2/157.1

256.7/239.1

342.1/324.1

The meta-bolites of six kinds of mixed probe substrates: Paracetamol (1 μ g/ml), 1-hydroxyl midazolam (1 μ g/ml), 4-hydroxytoluene sulphur butyl urea (1 μ g/ml), Levorphanol d-form (1 μ g/ml), hydroxyl Bupropion (1 μ g/ml), 4-hydroxyl mephenytoin (1 μ g/ml), marks bent concentration range and is 1,5,25,100,500,1000ng/ml.

(d) test kit specification sheets provides according to GraphPad Prism 5 Project softwares and calculates IC ₅₀whether the method for value, there is the risk of drug interaction according to the concentration range overall evaluation:

The relative activity being calculated under different concns drug effect by formula 1:

Erel（%）=Ci(n)/Ci(0)×100% （1）

In formula, Erel is relative activity, the meta-bolites growing amount that Ci (n) records for different concns medicine group, and Ci (0) is the meta-bolites growing amount of blank group.Take relative activity Erel as ordinate zou, and the Log value of drug level is X-coordinate, by GraphPad Prism 5 Software on Drawings, is suppressed curve and is calculated IC ₅₀value.IC ₅₀>50 μ mol/L explanation medicine is to CYP450 enzyme unrestraint effect, 10<IC ₅₀<50 μ mol/L explanation medicine is weak inhibitor, 1<IC ₅₀<10 μ mol/L explanation medicine is medium inhibitor, IC ₅₀<1 μ mol/L explanation medicine is potent inhibitor, IC ₅₀during <10 μ mol/L, show that medicine likely produces the risk of drug interaction when clinical application.

The advantage of test kit of the present invention and investigative technique method is the preparation of NADPH regeneration system rapidly, the preparation of different concns medicine, the method that adds of mixed substrates and hepatomicrosome all needs only according to product description, high-throughput and simple and convenient, even if neophyty operation also can complete experiment, the domestic high throughput testing test kit that there is no this type of, has filled up domestic blank, test kit of the present invention has significant Social benefit and economic benefit, once promote the sure demand that meets vast medicament research and development person.LC-MS/MS detection method provides detection method simple and reliable, highly sensitive and that error is little, and the mass spectrum of any type is substantially general, final IC ₅₀value is calculated fairly simple convenience, can evaluate the risk of drug interaction according to concentration range.This provides and has been easy to simple operations and test kit and research method efficiently for medicine early screening and clinical application repercussion study.

Accompanying drawing explanation

Fig. 1 screens CYP450 enzyme inhibitor medicine schema

Six kinds of meta-bolites color atlass of six kinds of meta-bolites screenings of Fig. 2

Fig. 3 KETOKONAZOL suppresses curve to the semilog of CYP3A4

Fig. 4 naphthyl alcohol suppresses curve to the semilog of CYP1A2

Fig. 5 Quinidine suppresses curve to the semilog of CYP2D6

Fig. 6 Tranylcypromine suppresses curve to the semilog of CYP2C19

Fig. 7 sulfaphenazole suppresses curve to the semilog of CYP2C9

Fig. 8 Isorhamnetol suppresses curve to the semilog of CYP2C9

Fig. 9 Isorhamnetol suppresses curve to the semilog of CYP1A2

Figure 10 Isorhamnetol suppresses curve to the semilog of CYP2C19

Embodiment 1

The restraining effect of positive inhibitor to rat CYP450 enzyme

1. experiment material

1.1 medicines and reagent

The detection kit that screening CYP450 enzyme suppresses, hydrochloric acid Pu Nailuoer and α-naphthoflavene, sulfaphenazole, Tranylcypromine, Quinidine, KETOKONAZOL are purchased from Products in China check calibrating institute, acetonitrile, methyl alcohol are purchased from Fisher(chromatographically pure), experimental water is Hangzhou WAHAHA group pure water.

1.2 key instruments and equipment

Liquid chromatograph/mass spectrometer

2. test method

2.1. incubation system

The detection kit preparation that reaction system suppresses according to screening CYP450 enzyme, includes NADPH regeneration system rapidly (2.5mmol/L NADPNa2,10U/mL glucose-6-phosphate dehydrogenase, 20mmol/L magnesium chloride, 50mmol/L G6P.), 0.1M phosphate buffered saline buffer, the rat liver microsomes of 20mg/ml and CYP enzyme mixed probe substrate (in Table 2-1), positive inhibitor series concentration (in Table 2-2), by above-mentioned regeneration system rapidly, medicine and probe substrate are mixed, after 37 ℃ of water-bath preincubate 5min, add rat liver microsomes (protein concentration 0.4mg/mL) to start reaction, each concentration is established 3 parallel sampleses, after 1h, use the stop buffer termination reaction that contains interior mark 100ng/ml (Proprasylyte), vortex 2min, 4 ℃, 14, the centrifugal 10min of 000 * g, get supernatant 10 μ L sample introduction LC-MS/MS, with the quantitative analysis method of the CYP enzyme mixed probe substrate utilization product of having set up, measure the generation of each probe substrate meta-bolites.By the growing amount of probe substrate meta-bolites, calculated the relative reactivity of CYP isozyme, by formula 1, calculate the relative activity under the positive inhibitor effect of different concns:

Erel（%）=Ci(n)/Ci(0)×100% （1）

In formula, Erel is relative activity, the meta-bolites growing amount that Ci (n) records for the positive inhibitor group of different concns, and Ci (0) is the meta-bolites growing amount of blank group.Take relative activity Erel as ordinate zou, and the Log value of positive inhibitor concentration is X-coordinate, by GraphPad Prism 5 Software on Drawings, is suppressed curve and is calculated IC ₅₀value.

Probe substrate and the meta-bolites of table 2-1.CYP isozyme

Table 2-2CYP enzyme suppresses rat liver microsomes and hatches positive inhibitor series concentration

3. the inhibition potential of positive inhibitor to CYP isozyme

In rat liver microsomes incubation system, evaluated the restraining effect of positive inhibitor to 5 main CYP activity of isoenzyme.Show 3-1 result and show, in 5 CYP isozymes, each positive inhibitor all has stronger restraining effect to CYP450 enzyme, and within the scope of the inhibition concentration requiring.

Each CYP enzyme hypospecificity inhibitor of table 3-1. IC ₅₀value

Embodiment 2

The restraining effect of Isorhamnetol to the different CYP450 enzymes of rat

1. experiment material

1.1 medicines and reagent

The detection kit that screening CYP450 enzyme suppresses, hydrochloric acid Pu Nailuoer and Isorhamnetol are purchased from Products in China check calibrating institute, acetonitrile, methyl alcohol are purchased from Fisher(chromatographically pure), experimental water is Hangzhou WAHAHA group pure water.

1.2 key instruments and equipment

Liquid chromatograph/mass spectrometer

2. test method

2.1. incubation system

The detection kit preparation that reaction system suppresses according to screening CYP450 enzyme, includes NADPH regeneration system rapidly (2.5mmol/L NADPNa2,10U/mL6-glucose phosphate dehydrogenase, 20mmol/L magnesium chloride, 50mmol/L6-glucose 1-phosphate1-.), 0.1M phosphate buffered saline buffer, the rat liver microsomes of 20mg/ml and CYP enzyme mixed probe substrate (in Table 4-1), Isorhamnetol series concentration (in Table 4-2), by above-mentioned regeneration system rapidly, medicine and probe substrate are mixed, after 37 ℃ of water-bath preincubate 5min, add rat liver microsomes (protein concentration 0.4mg/mL) to start reaction, each concentration is established 3 parallel sampleses, after 1h, use the stop buffer termination reaction that contains interior mark 100ng/ml (Proprasylyte), vortex 2min, 4 ℃, 14, the centrifugal 10min of 000 * g, get supernatant 10 μ L sample introduction LC-MS/MS, with the quantitative analysis method of the CYP enzyme mixed probe substrate utilization product of having set up, measure the generation of each probe substrate meta-bolites.By the growing amount of probe substrate meta-bolites, calculated the relative reactivity of CYP isozyme, by formula 1, calculate the relative activity under the effect of different concns Isorhamnetol:

Erel（%）=Ci(n)/Ci(0)×100% （1）

In formula, Erel is relative activity, the meta-bolites growing amount that Ci (n) records for different concns Isorhamnetol group, and Ci (0) is the meta-bolites growing amount of blank group.Take relative activity Erel as ordinate zou, and the Log value of Isorhamnetol concentration is X-coordinate, by GraphPad Prism 5 Software on Drawings, is suppressed curve and is calculated IC ₅₀value.

Probe substrate and the meta-bolites of table 4-1.CYP isozyme

Table 4-2CYP enzyme suppresses rat liver microsomes and hatches Isorhamnetol series concentration

3. the inhibition potential of Isorhamnetol to CYP isozyme

In rat liver microsomes incubation system, evaluated the restraining effect of Isorhamnetol to 6 main CYP activity of isoenzyme.Show 4-3 result and show, in 6 CYP isozymes, Isorhamnetol has medium tenacity restraining effect to CYP1A2,2C9 and 2C19, has potential drug interaction.

The inhibition IC of table 4-3. Isorhamnetol to each CYP isozyme ₅₀

Claims (10)

1. a test kit for high flux screening CYP450 enzyme inhibitor thing, it consists of: NADPH regeneration system rapidly; 0.1mol/L phosphate buffered saline buffer; Mixed probe substrate; The meta-bolites of mixed probe substrate; Hepatomicrosome; Described six kinds of mixed probe substrates are: Phenacetin (5mmol/L), midazolam (250 μ mol/L), tolbutamide (12mmol/L), Dextromethorphane Hbr (500 μ mol/L), Bupropion (2.5mmol/L), mephenytoin (5mmol/L); The meta-bolites of described six kinds of mixed probe substrates: Paracetamol (1 μ g/ml), 1-hydroxyl midazolam (1 μ g/ml), 4-hydroxytoluene sulphur butyl urea (1 μ g/ml), Levorphanol d-form (1 μ g/ml), hydroxyl Bupropion (1 μ g/ml), 4-hydroxyl mephenytoin (1 μ g/ml).

2. by the test kit of claim 1, it is characterized in that: NADPH regeneration system rapidly comprises A liquid: 2.5mmol/L NADPNa2,10U/mL6-glucose phosphate dehydrogenase, 20mmol/L magnesium chloride, B liquid: 50mmol/L6-glucose 1-phosphate1-.

3. by the test kit of claim 1, it is characterized in that: it is 29g Sodium phosphate dibasic that 0.1mol/L phosphoric acid buffer forms, 2.96g SODIUM PHOSPHATE, MONOBASIC, 42.5g sodium-chlor, 11.8g Repone K, is finally dissolved in 1 liter of distilled water.

4. by the test kit of claim 1, it is characterized in that: hepatomicrosome can be a kind of in the hepatomicrosome of mouse, rat, dog or monkey, and its concentration is 20mg/ml.

5. a research method for CYP450 enzyme inhibitor thing, is characterized in that comprising the steps:

1) by preparing the NADPH regeneration reaction system that 245 μ L hatch, comprise 50 μ L medicine series concentration+180, μ L mixed probe substrate+2.5, μ LB liquid+2.5, μ LA liquid+10 μ L0.1mol/L phosphoric acid buffer preincubate 5min in 37 ℃ of water-baths, add again the hepatomicrosome 5 μ L of 20mg/ml mouse, rat, dog or monkey, hatch 1h; Described 2.5 μ L mixed probe substrates are Phenacetin (50 μ mol/L) at 250 μ L incubation system final concentrations, midazolam (2.5 μ mol/L), tolbutamide (120 μ mol/L), Dextromethorphane Hbr (5 μ mol/L), Bupropion (25 μ mol/L), mephenytoin (50 μ mol/L);

2) then, be the proportional arrangement albumen precipitation liquid of methyl alcohol: acetonitrile=1:1 by volume, albumen precipitation liquid precipitate albumen with 3 times of volumes, vortex concussion 2 minutes, 14000 leave the heart 10 minutes, get supernatant sample introduction in LC-MS/MS, calculate the inhibition percentage of medicine series concentration to six kinds of enzymes, by GraphPad Prism 5 Software on Drawings, suppress curve and calculate IC ₅₀value.

6. by the research method of claim 5, it is characterized in that: in 250 μ L NADPH regeneration reaction system, NADPNa2 final concentration is 0.5mmol/L, the final concentration of glucose-6-phosphate dehydrogenase is 2U/mL, and the final concentration of magnesium chloride is 4mmol/L, and the final concentration of G6P is 10mmol/L.

7. by the research method of claim 5, it is characterized in that: medicine series concentration is 0.05,0.15,0.5,1.5,5,15,50,100 μ mol/L at 250 μ L incubation system final concentrations, add again a Control group, the organic phase of medicine dissolution is dissolved with acetonitrile, the ratio control of final whole incubation system acetonitrile is below 1%, and methyl alcohol or dmso solution organic phase ratio should be controlled at below 1 ‰.

8. by the research method of claim 5, it is characterized in that: the hepatomicrosome of mouse, rat, dog or monkey final concentration of protein in 250 μ L incubation systems is 0.4mg/ml.

9. by the research method of claim 5, it is characterized in that: the detection method of LC-MS/MS comprises:

1) liquid-phase condition: with mobile phase A (containing the pure water of 0.1% formic acid and 5mM ammonium formiate) and B(acetonitrile) gradient elution: 30%B(0min), 85%B(1.5-4.5min), 30%B(4.6min); 25 ℃ of column temperatures, flow velocity is 0.35mLmin ^-1, working time 5min; Sampling volume 10 μ L;

2) mass spectrum condition: in ESI source positive ion MRM mode, detect, 320 ℃ of capillary temperatures, capillary voltage+4000V, atomization voltage 25psi, dry gas flow velocity 10L/min, other mass spectroscopy parameter is in Table 1,

The ion pair of table 1 meta-bolites

Compound Paracetamol 4-hydroxytoluene sulphur butyl urea 4-hydroxyl mephenytoin Levorphanol d-form Hydroxyl Bupropion 1'-hydroxyl midazolam Ion pair 152.1/110.1 287/171.1 235.2/150.1 258.2/157.1 256.7/239.1 342.1/324.1

3) concentration of the meta-bolites of six kinds of mixed probe substrates: Paracetamol (1 μ g/ml), 1-hydroxyl midazolam (1 μ g/ml), 4-hydroxytoluene sulphur butyl urea (1 μ g/ml), Levorphanol d-form (1 μ g/ml), hydroxyl Bupropion (1 μ g/ml), 4-hydroxyl mephenytoin (1 μ g/ml), marks bent concentration range and is 1,5,25,100,500,1000ng/ml.

10. by the research method of claim 5, it is characterized in that: the relative activity being calculated under different concns drug effect by formula 1:

Erel（%）=Ci(n)/Ci(0)×100% （1）

In formula, Erel is relative activity, the meta-bolites growing amount that Ci (n) records for different concns medicine group, and Ci (0) is the meta-bolites growing amount of blank group.Take relative activity Erel as ordinate zou, and the Log value of drug level is X-coordinate, by GraphPad Prism 5 Software on Drawings, is suppressed curve and is calculated IC ₅₀value.IC ₅₀>50 μ mol/L explanation medicine is to CYP450 enzyme unrestraint effect, 10<IC ₅₀<50 μ mol/L explanation medicine is weak inhibitor, 1<IC ₅₀<10 μ mol/L explanation medicine is medium inhibitor, IC ₅₀<1 μ mol/L explanation medicine is potent inhibitor, IC ₅₀during <10 μ mol/L, show that medicine likely produces the risk of drug interaction when clinical application.