CN102898295A - UGT1A9/1A8 specific probe substrate and use thereof - Google Patents
UGT1A9/1A8 specific probe substrate and use thereof Download PDFInfo
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- CN102898295A CN102898295A CN2012103934038A CN201210393403A CN102898295A CN 102898295 A CN102898295 A CN 102898295A CN 2012103934038 A CN2012103934038 A CN 2012103934038A CN 201210393403 A CN201210393403 A CN 201210393403A CN 102898295 A CN102898295 A CN 102898295A
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Abstract
The invention discloses naphthoquinone compound alkannin, which can be used for quantitatively testing the enzyme activity of glucuronic acid transferase UGT1A9 and UGT1A8 as UGT1A9 and UGT1A8 specific probe substrate. The use is implemented by the following step of: selecting alkannin as a specific probe substrate, carrying out the UGT catalytic reaction of a specific substrate by using a UGT in-vitro incubation system, quantitatively testing the enzyme activity of UGT1A9 and UGT1A8 in biological samples and cells by quantitatively testing the lost amount of the substrate or the generated amount of the product. The substrate can be used for quantitative evaluation of the enzyme activity of UGT1A9/1A8 in different species, individuals and biological samples, calibration of propofol anesthetic metabolizing capacity of certain tissues, and calibration and estimation of the elimination rate of compounds which are mainly metabolized by UGT1A9 in specific organisms based on in-vitro metabolism dynamic data.
Description
Technical field
The invention belongs to medical technical field, be specifically related to specific probe substrate and the application thereof of a kind of UGT1A9/1A8.
Background technology
UGT(uridine diphosphate glucuronate transferring enzyme, Uridine diphosphate-glucuronosyltransferase) enzyme family is metabolic enzyme system important in the human body, it is responsible for removing multiple endogenous compound such as bilirubin, serotonin, thyroxine etc. and a large amount of exogenous compounds such as Irinotecan, thunder Lip river former times phenol, the medicines such as paracetamol, and the food such as stilboestrol, dihydroxyphenyl propane and environmental poisonous substance etc. (Drug Metabolism Disposition.2004,32:1201-1208).1 family in the UGT enzyme and 2 families are two major families of UGT enzyme system, and wherein UGT1 family has 9 hypotype: 1A1,1A3,1A4,1A5,1A6,1A7,1A8,1A9 and 1A10.9 hypotypes of UGT1A have obvious tissue specificity in the tissue distribution of human body.UGT1A1 wherein, 1A3,1A4,1A6,1A9 have abundant expression (1A6 and the 1A9 expression in kidney is also higher than liver) in liver, and 1A4,1A6 and 1A9 express in enteron aisle hardly.UGT1A7 is mainly in tissue distribution such as esophagus and tracheae and uterine neck, and UGT1A8 and 1A10 be expressed in abundance in small intestine and colon mainly, does not express in the liver.
The in-vitro evaluation system of UGT enzymic activity mainly comprises (the Toxicology in Vitro2006 such as the freshly extd Gan Xi of restructuring Dan Mei ﹑ mammiferous subcellular fraction Zu Fen ﹑ Bao ﹑ primary cultured hepatocyte, liver Qie Pian ﹑ liver perfusion, 135-153), wherein the single enzyme of commercial people's Tooth-Lid Factor T mainly comes from the clonal expression system that bacterium, insect cell, mammalian cell and yeast are set up, and subcellular components mainly comprises microsome, S9 composition.Adopt these standardized appraisement systems, in conjunction with corresponding cofactor (as: UDPGA etc.) and incubation conditions, can by metabolite clearance or the product formation rate of detection probes substrate, the UGT enzymic activity of expressing in the above-mentioned various systems be characterized.
A plurality of hypotypes among the UGT1A have very high homology at aminoacid sequence, thereby the substrate of catalysis has widely intercrossing, and the specific probe substrate of each hypotype of UGT lacks very much at present.UGT1A9 on aminoacid sequence with 1A7,1A8 has high homology (〉 93% between the 1A10 hypotype), thereby often show to have the metabolic characteristics of cosubstrate between these enzymes.Because the UGT1A9 hypotype mainly is distributed in liver and kidney, and 1A8,1A10 mainly expresses in intestines and do not express simultaneously (Plos One.2007.e396) in liver organization.So external take tissue particles body and S9 in main metabolism system, although a compound is the cosubstrate of UGT1A8 and UGT1A9, but because these two kinds of enzymes also can be resolved clear to this compound in the metabolic capacity of liver and enteron aisle UGT1A9 and UGT1A8 in the tissue distribution specificity of human body, if it not only not can be used as the specific probe substrate of UGT1A9 in the liver to this compound by other UGT enzymes metabolism simultaneously, can be used as again the specific probe substrate of enteron aisle UGT1A8.UGT1A9 mainly is responsible for some non-steroidal anti-inflammatory drugs of metabolite clearance such as paracetamol, indomethacin etc., her tolcapone of ancillary drug and some natural products such as the flavones for the treatment of parkinsonism, anthraquinone, coumarin kind compound etc., and UGT1A8 mainly is responsible for metabolism such as medicine thunder Lip river former times phenol, morphine, natural flavones etc.Assessment tool to the metabolic activity of these two enzymes is of great significance.
In sum, the screening of the specific probe substrate of UGT enzyme and develop significant.At present the anaesthetic Rapinovet be exactly the UGT1A9 metabolism be main medicine (it can be generated its single glucuronidation product by UGT1A9,1A7 and 1A8 catalysis), be widely used clinically.Among the present invention Shikonin have that meta-bolites single (only generating a single glucuronidation product), metabolic enzyme highly selective (in only UGT1A9 metabolism of liver, the intestines only UGT1A8 participate in its metabolism), metabolic rate are fast, the easy characteristics such as detection of meta-bolites.In addition, Shikonin is as the kasher additive of the countries such as China and Australia, and it has high security (the oral LD of mouse
50Be 2700-7990mg/kg), so its probe as UGT1A9 in the body is safer than anaesthetic Rapinovet.And enteron aisle main metabolic enzyme UGT1A8 does not have the report of specific probe substrate always, and proposition of the present invention will be filled up the blank in this field.
Summary of the invention
The purpose of this invention is to provide a kind of naphthoquinone compound Shikonin and application thereof, this Shikonin can be used as the external probe substrate of high specific of UGT1A9 and UGT1A8, and the enzyme that is used for quantitative assay different sources biological specimen UGT1A9 and UGT1A8 is lived.Owing to have not yet to see the specific probe substrate of bibliographical information UGT1A8, Shikonin can specific evaluation body as probe substrate or tissue in UGT1A9 enzyme and UGT1A8 enzyme to the disposing capacity of medicine, significant to the drug metabolism related basic research.In addition, Shikonin is compared the advantage of having more as the probe substrate of liver UGT1A9 with Rapinovet.
The invention provides a kind of naphthoquinone compound Shikonin, the name of this Shikonin is called 5,8-dihydroxyl-2-(1-hydroxy-4-methyl-3-pentenyl)-1,4-naphthoquinone, it is the specific probe substrate of uridine diphosphate glucuronate transferring enzyme 1A9 and 1A8, and its structure as shown in Figure 1.
The present invention also provides UGT1A8 or UGT1A9 enzyme method alive in the described Shikonin quantitative assay living things system, adopt Shikonin as probe substrate, the amount of cancellation by the substrate of detection by quantitative in the unit time or the growing amount of its O-glucuronidation product are measured the activity of UGT1A9 in each biological sample and the cell or 1A8 enzyme; Its measuring method is:
---in the system with Shikonin as the specific probe substrate; Concentration of substrate is selected between 1/5 ~ 5Km;
---in the incubation system with cofactor UDPGA, temperature of reaction is between 10 ℃ to 60 ℃;
---the reaction times is 5-120 minute, guaranteeing the corresponding glucuronidation meta-bolites of above substrate Shikonin list glucuronidation product (5, termination reaction when 8-dihydroxyl-2-(1-O glucuronic acid-4-methyl-3-pentenyl)-1,4-naphthoquinone) reaching quantitative limit and substrate conversion efficiency and be lower than 10%;
Substrate reduction or single glucuronidation product growing amount are as the evaluation index of uridine diphosphate glucuronate 1A9 and 1A8 enzymic activity in the analytical unit time.
Wherein detection substrate reduction or its quantitative detecting method to glucuronidation product growing amount are 517nm for adopting array diode detector maximum absorption wavelength, and mass spectrum, radio isotope tracer technique etc. also can be used for detecting Shikonin and glucuronidation product thereof.Used detector can be used one or more series connection wherein.
Shikonin provided by the invention be used for to be demarcated the ability of particular organization's metabolism anaesthetic Rapinovet, and to demarcate and estimate the UGT1A9 metabolism be that main compound is in the metabolic clearance rate of specific body.
Adopt human recombinant to express UGT1A9 or the single enzyme of 1A8, hepatomicrosome, kidney microsome or intestines microsome or corresponding S9 incubation system are investigated, pass through correlation analysis, specificity suppresses experiment, single enzymes metabolism of recombinating reacts, and the evidence of enzymatic reaction kinetics several respects, but proof Shikonin highly selective by liver UGT1A9 enzyme and enteron aisle UGT1A8 enzyme catalysis metabolism and generate Shikonin 2-(1-O)-single glucuronidation meta-bolites (as shown in Figure 3) UFLC collection of illustrative plates sees Fig. 4, the structure of meta-bolites is seen Fig. 4.
Specific probe substrate as UGT1A9 and 1A8, Shikonin can be used for detecting the activity of UGT1A9/1A8 in the single enzyme of people's Tooth-Lid Factor T, especially be suitable for UGT1A9 that bacterium, insect cell, mammalian cell and yeast clonal expression system are produced and the enzyme activity determination of UGT1A8 recombinase, and the activity of UGT1A9 and UGT1A8 is demarcated in the prepared products such as the microsome of multiple mammalian tissues organ origin, S-9.
Select Shikonin of the present invention to have following outstanding advantage as the external activity of the specific probe substrate detection enzyme of uridine diphosphate glucuronate transferring enzyme 1A9 and 1A8:
(1) highly selective: Shikonin can by specifically metabolism of the UGT1A9 in liver or the kidney, also can be generated by the UGT1A8 specific metabolic in the enteron aisle corresponding single glucuronidation product.
(2) be easy to obtain: the substrate Shikonin is the native chemical composition that contains in the Chinese Drug Zicao, and the source simply is easy to separation and purification, need not chemosynthesis.
(3) be easy to detect, Shikonin can utilize the array diode detector to detect, and the UV detector also has certain absorption to it, and these detectors relatively easily obtain.Rapinovet does not have spectral absorption can only adopt mass spectrometric detection or isotopic labeling because its molecular structure is simple, and testing cost is higher.
(4) high security: Shikonin is legal foodstuff additive, and it is compared with Rapinovet, and the LD50 value is higher, and security is better.
Description of drawings
Fig. 1. the chemical structural formula of Shikonin;
Fig. 2. the single enzyme screening experiment of the UGT of Shikonin;
Fig. 3. the UGT catalysis metabolism synoptic diagram of Shikonin;
Fig. 4. the metabolism spectrogram of Shikonin in people's hepatomicrosome;
Fig. 5. Shikonin 2-(1-O)-mass spectrum of single glucuronidation product.
Embodiment
The following examples will be further described the present invention, but not thereby limiting the invention.
Embodiment 1: Shikonin is used for the recombinate enzyme of single enzyme UGT1A9 of quantitative assay and lives
(1) prepares 180 μ l UGT metabolic reaction systems in advance, comprise Tris-HCl damping fluid (5mM), the 5mM MgCl of pH7.4
2, rhCC16 T1A9 (0.015mg/ml), Shikonin final concentration are 35 μ M, incubate in advance 5 minutes under 37 ℃ of conditions;
(2) in reaction system, add 20 μ l40mM UDPGA(final concentration 4mM) initial action; After (3) 20 minutes, add 100 μ l ice cold methanol, behind the concuss, termination reaction;
(4) use high speed freezing centrifuge at 4 ℃, under the condition of 20,000 * g, high speed centrifugation was got supernatant after 20 minutes, carried out UFLC-UV and detected;
(5) under 517nm, Shikonin and glucuronidation product thereof are carried out detection by quantitative.The maximum catalytic rate that calculates rhCC16 T1A9 enzyme is 150.3pmol/mim/mg.
Embodiment 2: Shikonin is used for the enzyme of quantitative assay people hepatomicrosome UGT1A9 and lives
(1) reaction system is the same.It is people's hepatomicrosome that UGT1A9 in the system is replaced, and MC protein concentration is 0.025mg/ml, and the reaction times is 20 minutes.
(2) 4 ℃, high speed centrifugation was got supernatant after 20 minutes under the condition of 20,000 * g, carried out UFLC-UV and detected;
(3) under 517nm, Shikonin and glucuronidation product thereof are carried out detection by quantitative.Can record that the maximum catalytic rate of UGT1A9 is 92.6pmol/min/mg in people's hepatomicrosome.
Embodiment 3: the enzyme that detects in the single enzyme of rhCC16 T1A8 is lived
Reaction conditions and operating process are identical with case study on implementation 1, and the metabolism that just replaces UGT1A9 to carry out Shikonin UGT1A8 is hatched.It is active to utilize identical method can determine different batches rhCC16 T1A8.The maximum speed of reaction of measuring the rhCC16 T1A8 of this experiment is 9.67pmol/min/mg.
Case study on implementation 4: the correlation analysis of Shikonin and propofol metabolism
Take Shikonin and Rapinovet as substrate, in 12 routine people liver individual specimen, carry out hatching of UGT metabolic reaction respectively, the reaction incubation conditions is with embodiment 1 and 2.Measure the generating rate of Shikonin/Rapinovet UGT meta-bolites in every routine people's liver microsomes incubation system, respectively take the generating rate of Shikonin/Rapinovet UGT meta-bolites as horizontal, ordinate zou mapping, formed loose point is carried out linear regression analysis, the r value of gained is 0.91, points out Shikonin and Rapinovet to have similar liver metabolism enzyme and close UGT1A9 metabolism contribution rate.
Claims (5)
1. naphthoquinone compound Shikonin, chemical name is 5,8-dihydroxyl-2-(1-hydroxy-4-methyl-3-pentenyl)-1, the 4-naphthoquinones, it is characterized in that: this Shikonin is as the specific probe substrate of uridine diphosphate glucuronate transferring enzyme 1A9 and 1A8, and its structure as the formula (1).
Formula (1)
Shikonin claimed in claim 1 be used for quantitative assay living things system UGT1A8 or
The method that the UGT1A9 enzyme is lived, it is characterized in that: adopt Shikonin as probe substrate, amount of cancellation by the substrate of detection by quantitative in the unit time or, 2-(1-O)-growing amount of glucuronidation product measures the activity of UGT1A9 in each biological sample and the cell or 1A8 enzyme.
3. the method for living according to UGT1A8 or UGT1A9 enzyme in the Shikonin quantitative assay living things system claimed in claim 2, it is characterized in that: the condition of described method is: selected concentration of substrate is between 1/5 ~ 5Km; Incubation system pH is between 5.5 ~ 10.5; Temperature of reaction is between 20 ~ 60 ℃; Product production rate or substrate conversion efficiency are lower than 10%.
4. the application of Shikonin claimed in claim 1, it is characterized in that: Shikonin is used for quantitative assay Tooth-Lid Factor T1A9, the UGT1A8 single enzyme as UGT1A9 and UGT1A8 specific probe substrate, animal liver, kidney and intestinal tissue microsome, the enzyme of UGT1A9 or UGT1A8 is lived among the S9.
5. the application of Shikonin claimed in claim 1 is characterized in that: Shikonin be used for to be demarcated the ability of particular organization's metabolism anaesthetic Rapinovet, and to demarcate and estimate the UGT1A9 metabolism be that main compound is in the metabolic clearance rate of specific body.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146804A (en) * | 2013-02-04 | 2013-06-12 | 中国科学院大连化学物理研究所 | Specificity probe zymolyte of glucuronic acid transferase UGT1A1 and application |
| CN104178554A (en) * | 2014-07-24 | 2014-12-03 | 暨南大学 | Application of oxymetazoline as special substrate for glucuronyl transferase UGT1A9 |
| CN104592986A (en) * | 2014-12-29 | 2015-05-06 | 大连理工常熟研究院有限公司 | Specific fluorescent probe for glucuronyl transferase UGT1A1 and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102211997A (en) * | 2011-03-28 | 2011-10-12 | 上海交通大学 | Alkannin naphthazarine mother nucleus acetoxylation derivative as well as preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102211997A (en) * | 2011-03-28 | 2011-10-12 | 上海交通大学 | Alkannin naphthazarine mother nucleus acetoxylation derivative as well as preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| SI-CHENG LIANG ET AL.,: "Determination of propofol UDPglucuronosyltransferase (UGT) activities in hepatic microsomes from different species by UFLC–ESI-MS", 《JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146804A (en) * | 2013-02-04 | 2013-06-12 | 中国科学院大连化学物理研究所 | Specificity probe zymolyte of glucuronic acid transferase UGT1A1 and application |
| CN103146804B (en) * | 2013-02-04 | 2015-04-22 | 中国科学院大连化学物理研究所 | Specificity probe zymolyte of glucuronic acid transferase UGT1A1 and application |
| CN104178554A (en) * | 2014-07-24 | 2014-12-03 | 暨南大学 | Application of oxymetazoline as special substrate for glucuronyl transferase UGT1A9 |
| CN104592986A (en) * | 2014-12-29 | 2015-05-06 | 大连理工常熟研究院有限公司 | Specific fluorescent probe for glucuronyl transferase UGT1A1 and application thereof |
| CN104592986B (en) * | 2014-12-29 | 2017-07-18 | 大连理工常熟研究院有限公司 | The specificity fluorescent probe of glucuronyl transferase UGT1A1 a kind of and its application |
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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 |
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