CN102993263B - Specific probe substrate for cytochrome P450 3A4 enzyme and application of substrate - Google Patents
Specific probe substrate for cytochrome P450 3A4 enzyme and application of substrate Download PDFInfo
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Abstract
The invention provides a specific probe substrate for a cytochrome P450 3A4 enzyme and an application of the substrate in CYP3A4 enzyme activity measurement. The specific operation flow of the enzyme activity measurement comprises the following steps: carrying out the CYP catalytic reaction of the specific substrate by virtue of a CYP in-vitro incubation system by selecting any monomer in toad steroid series of compounds as a high-specificity probe substrate; and measuring the activity of the CYP3A4 enzyme in each biological sample and each cell by quantitatively detecting a product generated quantity or a substrate eliminated quantity in a unit time. The specific probe substrate provided by the invention can be used for quantitative evaluation of CYP3A4 enzyme activity in biological samples belonging to different species and deriving from different individual sources, and quantitative measurement of CYP3A4 enzyme activity in animal tissue cell culture fluids and cell products deriving from different sources.
Description
Technical field
The invention belongs to medical art; be specifically related to application steroid vinyl compound; as Cinobufagin, remove acetyl Cinobufagin, resibufogenin and Toadpoison Medicine three compounds and analog thereof; it can be used as the Specific probe of Cytochrome P450 3A4 enzyme; for the quantitative assay that Cytochrome P450 3A4 enzyme in different sources biological specimen is lived, to realizing assessment important drugs metabolic enzyme CYP 3A4 being disposed to medicine ability.
Background technology
Cytochrome P 450 enzymes (Cytochrome P450, CYP) superfamily contains most important drug metabolism enzyme in body, and participate in the metabolism of about more than 80% marketed drug, the medicine wherein through the metabolism of Cytochrome P450 3A subfamily accounts for 50%.Have expressed four kinds of Cytochrome P450 3A hypotypes in human body, be respectively Cytochrome P450 3A4, P4503A5, P4503A7 and P4503A43.Cytochrome P450 3A4 accounts for 30 ~ 60%(Annu Rev Pharmacol Toxicol 1999 of total cytochrome P 450 enzymes content, 39:1 – 17), distribution is widely had in liver, intestines, kidney, spleen, each organ of heart, the Cytochrome P450 3A5 low compared to expression amount in crowd, expression frequency is low, is considered to the mono-enzyme of topmost Cytochrome P450 3A that adult is expressed always.In general, it is concrete that the enzyme of the various hypotypes in CYP3A subfamily all has similar substrate, and that is, if a certain substrate is by CYP3A4 catalyze metabolic, so this substrate is just most probably simultaneously by the enzymes metabolism of CYP3A5 and other CYP3A family.At present, that have been found that or in vitro, the CYP3A subfamily probe substrate of widespread use in In vivo study, as midazolam (midazolam), Nifedipine (nifedipine), testosterone (testosterone), estradiol (estradiol) etc., it is the specific substrate of CYP3A4 and CYP3A5 all simultaneously.Corresponding, utilize these probe substrate to be exactly the summation of the catalytic capability of each single enzyme of Cytochrome P450 3A subfamily to the assessment result reflection that cytochrome P 450 enzymes carries out.But, recent research finds, even suitable with the catalytic capability of Cytochrome P450 3A4 (the Drug Metab Dispos 2002 of the catalytic capability that Cytochrome P450 3A5 manifests in some individuality, 30:883-91), now utilize current existing probe then can obscure the catalytic capability of Cytochrome P450 3A4 and 3A5, thus cannot clearly analysis of cells cytochrome p 450 3A4 to the metabolite clearance ability of certain medicine.
In addition, mouse (Cytochrome P450 3A1 ﹑ Cytochrome P450 3A2), dog (Cytochrome P450 3A12, Cytochrome P450 3A26), rabbit (Cytochrome P450 3A6), pig (Cytochrome P450 3A29), in the mammalian bodies such as monkey (Cytochrome P450 3A64), also the isozyme of high expressing cell cytochrome p 450 3A4, the substrate between these enzymes has plyability widely.The high specific probe utilizing Cytochrome P450 3A4 also can realize evaluation to the isozyme of Cytochrome P450 3A4 in different genera and investigation in determination of activity aspect in vitro.
At present, the in-vitro evaluation system of Cytochrome P450 3A4 enzymic activity mainly comprises (the Toxicology in Vitro 2006 such as restructuring Dan Mei ﹑ mammiferous subcellular fraction Zu Fen ﹑ freshly extd Gan Xi Bao ﹑ primary cultured hepatocyte, liver Qie Pian ﹑ liver perfusion, 135 – 153), the clonal expression system that wherein the mono-enzyme of commercial reconstitution cell cytochrome p 450 3A4 mainly comes from bacterium, insect cell, mammalian cell and yeast are set up, and subcellular components mainly comprises microsome, cell cytosol and S9 composition.Adopt these standardized appraisement systems, in conjunction with corresponding cofactor (as: NADPH etc.) and incubation conditions, by metabolite clearance or the product formation rate of detection probes substrate, the Cytochrome P450 3A4 enzymic activity expressed in above-mentioned various system is characterized.The people such as Yu Chen (CN101308119) also utilize liver trace puncture method to construct novel micro hepatic tissue incubated in vitro system, and adopt midazolam as probe substrate, by measuring the ratio of midazolam and its meta-bolites 1-hydroxymidazolam content, be used as Cytochrome P450 3A external activity evaluation index.
In sum, assessment Cytochrome P450 3A4 metabolic enzyme activity, key is the probe medicament selecting high specific.
Summary of the invention
The present invention relates to application steroid vinyl compound and it is in the purposes detected in Cytochrome P450 3A4 enzymic activity and detection method thereof, bufadienolide biotransformation compounds (bufadienolides) is a class Analysis of Steroids, this series compound has the basic structure shown in Fig. 1, it can be used as the high specific probe substrate of Cytochrome P450 3A4 enzyme, lives for CYP3A4 enzyme in quantitative assay different sources biological specimen.Different from the Cytochrome P450 3A probe/standard substrate reported at present, the monomer in bufadienolide biotransformation class series compound has the ability that high specific characterizes Cytochrome P450 3A4 enzymic activity, and it is not by CYP3A5 catalysis.Therefore, it is possible to the evaluation body of high specific or tissue in CYP3A4 enzyme to the disposing capacity of medicine, significant to external drug metabolism study.
The present invention specifically provides the Specific probe of a cytochromoid P450 3A4 enzyme, it is characterized in that: the Specific probe of described Cytochrome P450 3A4 enzyme has the monomer in the bufadienolide biotransformation class series compound of structure shown in Fig. 1, wherein R for a class
1for-H ,-OH or-OAc group.The preferred Cinobufagin of monomer wherein in bufadienolide biotransformation class series compound, remove acetyl Cinobufagin, resibufogenin or Toadpoison Medicine.
Present invention also offers the application of described Cytochrome P450 3A4 enzyme spcificity probe substrate, it is characterized in that: by the one in bufadienolide biotransformation class series compound monomer as Specific probe, the reduction of the growing amount or substrate that measure corresponding meta-bolites by quantitative timing realizes the detection of Cytochrome P450 3A4 enzymic activity.
Adopt reconstitution cell cytochrome p 450 list enzyme, liver microsomes incubation system is investigated, pass through correlation analysis, specificity Inhibition test, single enzymes metabolism of recombinating reacts, and the evidence of enzymatic reaction kinetics several respects, prove Cinobufagin (cinobufagin, CB), resibufogenin (resibufagenin, and Toadpoison Medicine (bufalin RB), BF) three compounds all can be specific through Cytochrome P450 3A4 enzymes metabolism (as Suo Shi Fig. 2 ~ 4), generate 5 beta-hydroxy Cinobufagins (he is clever for magnificent toadpoison) successively, 1a-hydroxylation Cinobufagin, 5 beta-hydroxy esters bufotalins (marinobufagin) and 5 beta-hydroxy Toadpoison Medicine (Cinobufagin far away), Fig. 5 ~ 7 are shown in by UFLC collection of illustrative plates, the structure of meta-bolites is shown in Fig. 8.The metabolic evaluation systems such as the various mammiferous freshly extd Gan Xi Bao ﹑ primary cultured hepatocyte of further employing, liver Qie Pian ﹑ liver perfusion are investigated, and find that this metabolic reaction has very good specificity.
As the probe substrate of the Cytochrome P450 3A4 of high specific; Cinobufagin, go the arbitrary monomer in the bufadienolide biotransformation compounds such as acetyl Cinobufagin, resibufogenin, Toadpoison Medicine all can be used for detecting Cytochrome P450 3A4 enzymic activity; be especially suitable for the enzyme activity determination to the Cytochrome P450 3A4 recombinase that bacterium, insect cell, mammalian cell and yeast clonal expression system are produced, and in the prepared product such as microsome, S-9 of multiple mammalian tissues organ origin, the activity of CYP 3A4 is demarcated.
The application of the Specific probe of described Cytochrome P450 3A4 enzyme provided by the invention, namely adopts described probe substrate to detect Cytochrome P450 3A4 enzymic activity, its measuring method and condition as follows:
A. select one in bufadienolide biotransformation class series compound monomer as Specific probe; Concentration of substrate is 1-500 μM;
B. under the buffer solution system of pH=7.0 ~ 8.5, add the incubation system of NADPH or its generation structure, temperature of reaction is between 20 ~ 60 DEG C;
C. the reaction times is 10-120 minute, guarantee the corresponding hydroxylation metabolism product of above substrate reach quantitative limit and substrate conversion efficiency lower than 10% time termination reaction;
D. in the analytical unit time substrate reduction or single hydroxylated product growing amount as the evaluation index of Cytochrome P450 3A4 enzymic activity.
Wherein the quantitative detecting method of detection substrate reduction or its corresponding hydroxylated product growing amount is ultra-violet absorption spectrum, mass spectrum, radio isotope tracer technique, fluorescence excitation spectrum, evaporat light scattering or electrochemistry spectral detection.All detectors comprise one or more series connection in ultra-violet absorption spectrum detector, mass spectrum, radio isotope tracer technique, evaporat light scattering or electrochemistry spectroscopic detector.
Select the Specific probe of Cytochrome P450 3A4 enzyme of the present invention to detect Cytochrome P450 3A4 enzyme external activity and there is following outstanding advantage:
(1) high specific, the arbitrary monomer in bufadienolide biotransformation class series compound all can be become corresponding single hydroxylated product by target cell cytochrome p 450 3A4 metabolism with high specificity.
(2) substrate Cinobufagin, resibufogenin, Toadpoison Medicine and corresponding meta-bolites thereof China his spirit of toadpoison, marinobufagin, Cinobufagin far away are all the native chemical compositions that the Chinese medicine dried venom of toads contains, and originate and are simply easy to separation and purification, without the need to chemosynthesis.
(3) detect sensitive, the compound with bufadienolide biotransformation class mother nucleus structure all has good ultraviolet absorption characteristic (295 ~ 300 nm) and ionising effect, can be convenient quantitative to it by routine analysis means.In clinical application, without the need to buying expensive test set separately.
Accompanying drawing explanation
Fig. 1. the basic structure formula of bufadienolide biotransformation compounds;
Fig. 2. the people of Cinobufagin recombinates single enzyme shaker test result;
Fig. 3. the people of resibufogenin recombinates single enzyme shaker test result;
Fig. 4. the people of Toadpoison Medicine recombinates single enzyme shaker test result;
Fig. 5. Cinobufagin (CB) and hydroxylation metabolism product (1-HCB, 5-HCB) color atlas in people's hepatomicrosome thereof;
Fig. 6. resibufogenin (RB) and hydroxylation metabolism product (5-HRB) color atlas in people's hepatomicrosome thereof;
Fig. 7. Toadpoison Medicine (BF) and hydroxylation metabolism product (5-HBF) color atlas in people's hepatomicrosome thereof;
Fig. 8. the hydroxylation metabolism path of the CYP3A4 mediation of Cinobufagin (CB), resibufogenin (RB) and Toadpoison Medicine (BF).
Embodiment
The following example is to further illustrate the present invention, instead of will limit its scope.
Embodiment one
cinobufagin is lived for the enzyme detecting people recombinant C YP3A4 and CYP3A4+ cytochrome b5 system
Utilize Cinobufagin to detect two kinds of people to recombinate single enzyme (in recombinant expression system containing cytochrome b5 and do not contain cytochrome b5 two kinds) difference in catalytic activity, concrete steps are as follows:
In (1) 200 microlitre In vitro metabolism reaction system, 10 mM G-6-Ps, the glucose-6-phosphate dehydrogenase (G6PD) of 1 unit, and 4 mM MgCl
2, the recombinant C YP3A4 concentration containing cytochrome b5 is 0.05 mg/ml, and Cinobufagin final concentration is 30 μMs, under 38 DEG C of conditions, incubate 5 minutes in advance;
(2) in reaction system, add 20 μ l NADP
+(final concentration 1 mM) initial action;
After (3) 10 minutes, add 200 μ l methyl alcohol, after concuss, termination reaction;
(4) adopt high speed freezing centrifuge, 20, under the condition of 000 × g, the above-mentioned system of high speed centrifugation, after 10 minutes, gets supernatant, carries out UFLC-UV and detects analysis;
(5) to operate the determination of activity of the recombinant C YP3A4 carried out not containing cytochrome b5 equally.
Under 296 nm, carry out detection by quantitative by ultra-violet absorption spectrum to the residual volume of Cinobufagin, representative UFLC-UV spectrogram as shown in Figure 5.The enzymic activity in the recombinant C YP3A4 not containing cytochrome b5 that the enzymic activity of the recombinant C YP3A4 containing cytochrome b5 utilizing Cinobufagin to record as probe substrate is compared improves 22.7%.
Embodiment two
resibufogenin is used for the mensuration that in 12 routine individuals hepatomicrosomes, CYP3A4 enzyme is lived
Buy the people hepatomicrosome sample of business-like 12 examples from Different Individual, the enzyme utilizing resibufogenin to measure CYP3A4 in people liver sample is lived, and concrete operations flow process is as follows:
In (1) 200 microlitre In vitro metabolism reaction system, 10 mM G-6-Ps, the glucose-6-phosphate dehydrogenase (G6PD) of 1 unit, and 4 mM MgCl
2, people's hepatomicrosome concentration is 0.2 mg/ml, and resibufogenin final concentration is 30 μMs, under 37 DEG C of conditions, incubate 3 minutes in advance;
(2) in reaction system, add 20 μ l NADP
+(final concentration 1 mM) initial action;
After (3) 10 minutes, add 200 μ l methyl alcohol, after concuss, termination reaction;
(4) adopt high speed freezing centrifuge, 20, under the condition of 000 × g, the above-mentioned system of high speed centrifugation, after 10 minutes, gets supernatant, carries out UFLC-UV and detects analysis;
Under 300 nm, carry out detection by quantitative by ultra-violet absorption spectrum to the growing amount of resibufogenin hydroxylated product, representative UFLC-UV spectrogram as shown in Figure 6.Measurement result shows, the formation speed according to resibufogenin meta-bolites has bigger difference in people's hepatomicrosome of different sources, and most high reactivity and lowest activity gap reach 5.6 times.
Embodiment three
the mensuration that the enzyme that Toadpoison Medicine is used for CYP3A4 in human liver cancer cell BEL7402 is lived
(1) incubate liquid by BEL7402 cell dilution by liver cell temperature, be placed in 6 well culture plates, every hole 4ml, puts into metal bath vibrator, 80 r/min, and 40 DEG C of continuous oscillations hatch 120 minutes;
(2) in culture plate, add Toadpoison Medicine, final concentration is 50 μMs;
Draw temperature after (3) 30 minutes and incubate liquid 200 μ l in-80 DEG C of Ultralow Temperature Freezer stopped reactions;
(4) sample adds 200ul methanol extraction albumen, and adopts high speed freezing centrifuge, and 20, under the condition of 000 × g, the above-mentioned system of high speed centrifugation, after 10 minutes, gets supernatant, carries out UFLC-ESI-MS and detects analysis
Adopt UFLC-ESI-MS to detect Toadpoison Medicine and meta-bolites thereof respectively, select the molecular ion peak [M+H] of SIM mode detection Toadpoison Medicine and meta-bolites thereof
+(
m/z387 &
m/z403).Result shows, the sensitivity utilizing UFLC-ESI-MS to detect Toadpoison Medicine and meta-bolites thereof can reach 0.1 nM, the activity of Cytochrome P450 3A4 enzyme in quantitative assay zooblast that can be sensitive.
Embodiment four
acetyl Cinobufagin is gone to live for the enzyme detecting CYP3A4 in people's intestines S9
Utilize Cinobufagin to detect the catalytic activity difference of CYP3A4 in two kinds of white people and yellow's intestines S9, concrete steps are as follows:
In (1) 200 microlitre In vitro metabolism reaction system, 10 mM G-6-Ps, the glucose-6-phosphate dehydrogenase (G6PD) of 1 unit, and 4 mM MgCl
2, be 0.2 mg/ml containing people's intestines S9(final concentration of protein), go acetyl Cinobufagin final concentration to be 50 μMs, under 40 DEG C of conditions, incubate 5 minutes in advance;
(2) in reaction system, add 20 μ l NADP
+(final concentration 1 mM) initial action;
After (3) 30 minutes, add 200 μ l methyl alcohol, after concuss, termination reaction;
(4) adopt high speed freezing centrifuge, 20, under the condition of 000 × g, the above-mentioned system of high speed centrifugation, after 10 minutes, gets supernatant, carries out UFLC-UV and detects analysis;
By ultra-violet absorption spectrum under 296 nm to going the residual volume of acetyl Cinobufagin to carry out detection by quantitative, comparatively yellow is high by 27% to find to live the enzyme of CYP3A4 in white people intestines S9.
Claims (1)
1. the method utilizing probe substrate to measure Cytochrome P450 3A4 enzymic activity, described probe substrate is for having the Toadpoison Medicine of formula (1) structure, it is characterized in that: employing Toadpoison Medicine is probe substrate, the activity of Cytochrome P450 3A4 enzyme is measured by substrate reduction in the quantitative assay unit time or single hydroxylated product growing amount, wherein:
A. concentration of substrate is 1-500 μM;
B. under the buffer solution system of pH=7.0 ~ 8.5, add the incubation system of NADPH or its generation structure, temperature of reaction is between 20 ~ 60 DEG C;
C. the reaction times is 10-120 minute, guarantee the corresponding hydroxylation metabolism product of above substrate reach quantitative limit and substrate conversion efficiency lower than 10% time termination reaction;
D. in the analytical unit time substrate reduction or single hydroxylated product growing amount as the evaluation index of Cytochrome P450 3A4 enzymic activity,
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| CN104892563B (en) * | 2014-03-06 | 2017-09-22 | 中国科学院大连化学物理研究所 | A kind of cromoci YP3A4 enzyme spcificitys probe reaction and its application |
| WO2015179999A1 (en) * | 2014-05-30 | 2015-12-03 | 中国科学院大连化学物理研究所 | Ratio-type fluorescent probe substrate of cytochrome oxidase cyp1a and use thereof |
| CN108796035B (en) * | 2018-05-31 | 2021-07-16 | 四川大学 | Specific probe reaction of cytochrome CYP3A7 enzyme and application thereof |
| CN114478383B (en) * | 2022-02-25 | 2024-03-01 | 上海中医药大学 | Fluorogenic substrate for detecting human cytochrome P4503A4 and preparation method and application thereof |
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Effective date of registration: 20180301 Address after: 215600 A 207 room A building center of Zhangjiagang Free Trade Zone, Suzhou Free Trade Zone, Jiangsu Patentee after: Zhangjiagang Institute of industrial technology, Dalian Institute of Chemical Physics, China Academy of Sciences Address before: 116023 Zhongshan Road, Liaoning, No. 457, Patentee before: Dalian Institute of Chemical Physics, Chinese Academy of Sciences |