KR102138548B1 - Composition for Assaying Activity of Uridine 5'-diphosphoglucuronosyltransferase Comprising Licoricidin and Use Thereof - Google Patents

Abstract

The present invention relates to a composition for evaluating uridine 5`-diphosphoglucuronosyltransferase activity including lycoricidine and a method for evaluating the activity of the enzyme of a new drug candidate using the same. When the composition according to an embodiment of the present invention is used, uridine 5`-diphosphoglucuronosyltransferase activity including UGT1A9, a biphasic metabolic enzyme of a candidate substance necessary for drug evaluation during the course of developing a new drug candidate substance, It can be effectively measured and used to predict drug interactions in the body.

Description

Composition for Assaying Activity of Uridine 5'-diphosphoglucuronosyltransferase Comprising Licoricidin and Use Thereof}

The present invention relates to a composition for evaluating uridine 5`-diphosphoglucuronosyltransferase activity including lycoricidine and a method for evaluating the activity of the enzyme of a new drug candidate using the same.

Licorice root has long been widely used as a herbal medicine and natural sweetener [1]. Licorice is a name derived from the roots and creeping stems of the Glycyrrhiza species, and contains various types of active ingredients with anti-cancer, anti-mutation, anti-viral and antibacterial effects [2-5]. The main components of licorice are triterpene saponins (glycyrrhizin), flavonoids (lycoflavonol, lycoricidine and lycoisoflavanone) and coumarin (isoglycicumaline) [6]. Among them, lycorisidine is a major prenylated isoflavone included in Glycyrrhiza uralensis Fisch. (Leguminosae), and has been reported to have pharmacological effects such as anti-transition, antioxidant and anti-genotoxicity [5, 7-9]. Although many studies have been conducted on the pharmacological effect of ricolithin, little research has been conducted on the metabolism of ricoritin in the human body.

Uridine 5'-diphosphoglucuronosyltransferase (UGT, EC 2.4.1.17) transfers glucuronic acid to a hydroxyl, carboxyl or amino group of a lipophilic compound to increase hydrophilicity. It is known to be one of the most important non-P450 enzymes that play an important role in drug metabolism [10]. In addition, UGT is involved in the regulation of biological activity of various intrinsic or extrinsic compounds. In particular, flavonoids having multiple hydroxyl groups are the most common substrates in which glucuronyl groups are conjugated by UGT. Representatively, quercetin is a typical antioxidant flavonoid widely distributed in vegetables, whose pharmacological effect is derived from the binding of glucuronide [11-13].

The present inventors studied metabolic pathways affecting lycoricidine in pooled HLM using high resolution quadrupole-orbitrap mass spectrometry. Although flavonoid glucuronation by UGT is important for bioactivity, lycolicidin binding in the human body has not been previously reported. Accordingly, the present inventors completed the present invention by revealing that lycolycidine is a new substrate of human UGT1A9 through a study on the metabolic profiling of lycoricidine in pooled HLM.

Republic of Korea Registered Patent No. 10-1735185

One aspect of the present invention is to provide a composition for evaluating the activity of uridine 5`-diphosphoglucuronosyltransferase containing lycoricidine.

Another aspect of the present invention is to provide a kit for evaluating uridine 5`-diphosphoglucuronosyltransferase activity comprising the composition.

Another aspect of the present invention is to provide a method for evaluating uridine 5`-diphosphoglucuronosyltransferase activity of a new drug candidate using the composition.

One aspect of the present invention provides a composition for evaluating the activity of uridine 5'-diphosphoglucuronosyltransferase (UGT) containing uriricidine.

Drug metabolism can occur in almost all organs (eg, intestine, lungs, and kidneys), but major drug metabolizing enzymes are expressed at high levels in the liver, making the most important organs for drug metabolism It can be called liver. The metabolic process is a process of increasing the water solubility of absorbed exogenous substances and converting it into an easily excreted form, which is an important defense system that prevents the accumulation of drugs in the body. However, there are cases in which the drug metabolic process is incomplete, and the toxicity increases in the metabolic process, so evaluating the activity of the drug metabolizing enzyme of the new drug candidate is an important part in the development of new drugs.

In general, the drug metabolic reaction is divided into a phase 1 (Phase I) and a phase 2 (Phase II) reaction, and the phase 1 reaction is typically a functional group (functional group), for example, a hydroxyl group, etc., to introduce the polarity of the substance ( It is a reaction that increases polarity, and the two-phase reaction is a conjugation reaction that occurs on the metabolite generated after the parent compound itself or the parent compound is introduced through a one-phase reaction to a polar functional group. Increase.

In particular, two-phase reactions include glucuronidation, sulfonation, glutathione conjugation, amino acid conjugation, methylation and acetylation reactions. And all reactions other than methylation and acetylation are reactions to increase water solubility, and uridine 5'-diphosphoglucuronosyltransferase (UTG) and its enzymes are two-phase metabolites. It is a representative enzyme.

Uridine 5`-diphosphoglucuronosyltransferase is a glycosyltransferase present in the cytoplasm, which delivers the glucuronic acid component of UDP-glucuronic acid to hydrophobic molecules. As an enzyme that acts as a catalyst, it mediates a glucuronidation reaction. Recently, new drug candidates that are being developed must identify the enzymes of UGT that are involved with glucuronylation by UGT, and to identify interactions with UGT enzymes by new drug candidates Selective substrates should be used to measure the activity of the enzyme.

On the other hand, the human UGT1A9 enzyme is one of nine UGT1A isozymes, and is mainly expressed in the liver and kidney [15, 16]. UGT1A9 is one of the physiologically and pharmacologically important UGT1A coenzymes and has a broad spectrum of substrates with large phenols such as dietary components, steroids and fatty acids [17]. In addition, UGT1A9 can metabolize various prescription drugs, including fibric acid-based drugs such as propofol, mycophenolic acid, fenofibric acid, and irinotecan, anticancer drugs, nonsteroidal anti-inflammatory drugs, and antiarrhythmic drugs [18-22].

According to one embodiment of the present invention, the uridine 5`-diphosphoglucuronosyltransferase may be one or more selected from the group consisting of UGT1A9, UGT2B7 and UGT1A3, but most preferably UGT1A9. That is, in the present invention, it was confirmed that lyricidine was selectively glucuronylated by UGT1A9, and higher reactivity was observed compared to mycophenolic acid, which is a substrate of the known UGT1A9, and UGT copper other than UGT1A9 It was confirmed that the activity of the enzyme was not reduced.

Other aspects of the invention include lycolicidine, glucose-6-phosphate, β-NADP+ and glucose-6-phosphate dehydrogenase and UDP-glucuronic acid. (UDP-glucuronic acid) containing a uridine 5`- diphospho glucuronyl transferase activity kit for evaluation.

In order to discover new drug candidates that are stable in drug metabolism, a metabolic stability test is performed. Since drug metabolism mainly occurs in the liver, the composition according to an embodiment of the present invention is a tissue fraction derived from the liver, for example, microsomes. It is preferable to further include (microsomes), cytosol or S9 fraction or primary hepatocyte. In addition, the composition may be prepared in the form of a kit, wherein, in order to generate NADPH required to provide a reducing power for catalytic reaction of a substrate of UGT in addition to lyricidine, NADPH can be generated in the kit. Any set of compounds can be used, but preferably, they include glucose-6-phosphate, β-NADP+ and glucose-6-phosphate dehydrogenase. It is preferred. In addition, UDP-glucuronic acid may be included to mediate the glucuronidation reaction of UGT, and additionally, alamethicin and β-glucuronidase inhibitor ( β-glucuronidase inhibitor).

Another aspect of the invention

a) contacting a new drug candidate substance to a composition comprising lycoridine;

b) incubating by adding glucose-6-phosphate, β-NADP+ and glucose-6-phosphate dehydrogenase to the result of step a);

c) culturing by adding UDP-glucuronic acid to the product of step b);

d) performing mass spectrometry of the culture result of step c); And

e) It provides a method for evaluating the activity of uridine 5`-diphosphoglucuronosyltransferase of a new drug candidate substance by confirming the presence or absence of monoglucuronide lyricidine from the results of the mass spectrometry.

Hereinafter, the method will be described in detail.

The method according to an embodiment of the present invention first performs a step of contacting a new drug candidate substance with a composition comprising the lycolicidine.

Richoricidine is a composition for evaluating the activity of one or more, most preferably UGT19A selected from the group consisting of UGT1A9, UGT2B7 and UGT1A3 in a two-phase reaction, to which a new drug candidate is added to the composition to perform preliminary culture. do.

Then, the method according to an embodiment of the present invention b) glucose-6-phosphate (glucose-6-phosphate), β-NADP+ and glucose-6-phosphate dehydrogenase (glucose-6) -phosphate dehydrogenase) to incubate; The step of culturing by adding UDP-glucuronic acid to the product of step b) is performed.

According to one embodiment, NADPH is required to provide a reducing power for the catalytic reaction of the UGT enzyme substrate, and as a set of compounds generating the NADPH, glucose-6-phosphate, β-NADP+, and Glucose-6-phosphate dehydrogenase is added, and UDP-glucuronic acid is added for glucuronylation by UGT.

Then, the method according to an embodiment of the present invention is subjected to a step of performing mass spectrometry of the culture result of step c).

The culture product of step c) is an enzyme reaction termination compound, for example. By adding acetonitrile, the reaction can be terminated and the supernatant obtained through centrifugation can be used.

Finally, the method according to an embodiment of the present invention, e) from the results of the mass spectrometry can be performed to determine the presence or absence of monoglucuronide lyricidine.

According to one embodiment, the mass spectrometry can be achieved by various mass spectrometry methods known in the art, but most preferably by LC-MS/MS. The evaluation is determined by confirming whether the new drug candidate substance inhibits the catalytic reaction of one or more, most preferably UGT19A, selected from the group consisting of UGT1A9, UGT2B7 and UGT1A3 of lyricidine.

When the composition according to an embodiment of the present invention is used, uridine 5`-diphosphoglucuronosyltransferase activity including UGT1A9, a biphasic metabolic enzyme of a candidate substance necessary for drug evaluation during the course of developing a new drug candidate substance, It can be effectively measured and used to predict drug interactions in the body.

1 is a schematic view showing a method of isolating lycoridine from the roots of Glycyrrhiza uralensis .
Figure 2 is a graph showing the metabolic stability of lyricidine through glucuronation of lyricidine in the Phase I and Phase II metabolic systems.
Figure 3 shows the results of the extracted ion chromatograms (EIC) of lyricidine and M1. In the presence of NGS, 50 μM of lycoricidine was incubated with pooled HLM at 37° C. for 1 hour, and (A) was confirmed with or without UDPGA (top) or (B) UDPGA. It is the result confirmed in the state including.
FIG. 4 is a diagram showing the CID spectrum of protonated lyricidine.
5 is a diagram showing the MS ² spectrum for monoglucuronylation.
A in FIG. 6 is a graph showing the production of time- and protein-dependent M1, and B is a graph showing the relative formation of M1 in human recombinant cDNA-expressed UGT isoenzyme.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are intended to illustrate one or more embodiments by way of example, and the scope of the present invention is not limited to these examples.

1. Experimental method

1.1. Compound and reagent

Lycorisidine is Glycyrrhiza by the method disclosed in FIG. It was isolated from the roots of uralensis . 10.0 kg of dried licorice root was refluxed 3 times with 10 L of methanol for 3 hours at 60°C. Thereafter, the methanol fraction of 3.3 kg obtained by vacuum concentration was suspended with distilled water, and separated into organic solvent fractions, that is, normal hexane, ethyl acetate, butanol fraction, and water layer. Among them, eight fractions (GU-1 to GU-8) were obtained by performing silica gel column chromatography, eluting 1.1 kg of ethyl acetate fraction by increasing the polarity of the mixed solution of methylene chloride and methanol. Thereafter, 151.7 g of fraction GU-5 was again subjected to silica gel column chromatography eluting with a mixture of methylene chloride and methanol (10% to 100%) to obtain four fractions (GU5-1 to GU5-4), A reverse phase column chromatography of 24.0 g of fraction GU5-2 eluting with a 40% to 80% methanol solution was performed to finally separate 3.2 g of lycolycitin.

Pooled Human Liver Microsomes, mixed gender, 20 mg/ml, hereinafter referred to as HLM) were purchased from Xenotech LLC (Lenexa, Kansas, USA), purified human recombinant cDNA-expressed UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7 Corning It was purchased from Gentest (Woburn, MA, USA). Reduced form of β-Nicotinamide adenine dinucleotide phosphate (β-NADPH), glucose 6-phosphate, glucose 6-phosphate dehydrogenase (glucose 6-phosphate dehydrogenase, uridine 5'-diphosphoglucuronic acid (UDPGA), alamethicin, and 1,4-saccharolactone, respectively, are Sigma ( St. Louis, Mo, USA), and all compounds used in this experiment in addition to the above compounds were used as products of analytical grade. For LCMS grade water and acetonitrile (ACN), Fischer Scientific (Pittsburgh, PA, USA) was used.

1.2. script Stability study

The metabolic stability study of lycoricidine was performed at two concentrations (10 μM and 50 μM) by separating lycolicidine from the roots of Glycyrrhiza uralensis according to the previously described method [8]. First, 0.5 mg/ml alameticin was treated with 0.25 mg/ml HLM and stored on ice for 15 minutes to activate the enzyme. Then, 0.1 M of potassium phosphate buffer (potassium phosphate buffer, pH 7.4) and 10 μM or 50 μM of lycoricidine were added and pre-incubated at 37° C. for 5 minutes. NADPH-generating system (hereinafter, NGS) solution (0.1 M glucose 6-phosphate, 10 mg/ml β-NADPH, and 1.0 U/ml glucose-6-phosphate dehydrogenase) was added and the reaction mixture was added to 10 μM uridine 5 Incubated at 37°C for 60 minutes in the presence of'-diphosphoglucuronic acid (UDPGA)'. Each hour, 50 μl of the reaction mixture was transferred to a new tube containing 50 μl of acetonitrile containing 0.1% formic acid to terminate the reaction. Thereafter, the mixture was vortexed for 15 seconds, centrifuged at 12,000 × g for 10 minutes, and then the supernatant was transferred to a glass vial and LC-MS analysis was performed.

1.3. In human liver microsomes, O -Glucuronyl-binding confirmation

After activating the enzyme by treating alameticin (0.5 mg/ml) in HLM (1 mg/ml), 50 μM lycoricidine was added to the mixture together with 0.1 M potassium phosphate buffer (pH 7.4) at 37° C. Pre-incubation for 5 minutes. The reaction was initiated by adding 10 μM UDPGA and NGS solution, and then further incubated at 37° C. for 60 minutes, and then the reaction was terminated by adding 400 μM of 100% ACN. The reaction mixture was vortexed for 15 seconds and then centrifuged for 10 minutes under conditions of 12,000 x g . Then, 550 μl of the supernatant was taken and completely evaporated using SpeedVac, and then stored at -80°C. The residue was dissolved in 100 μl of 20% MeOH with 0.1% formic acid, vortexed and centrifuged (12,000 × g , 10 min, 4° C.), then 10 μl of the supernatant was transferred to a glass vial and LC- Injection into C18 column for MS analysis.

1.4. Reaction phenotyping in recombinant human UGT

To identify UGT isoenzymes likely to be involved in the metabolism of lycoricidine, six purified recombinant human liver UGT coenzymes at 0.5 mg/ml in a reaction volume of 200 μl in the presence of NGS and UDPGA (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, and UGT2B7) were incubated with 10 μM of lycolicidine at 37° C. for 60 minutes, respectively, and the reaction was terminated by adding 400 μl of 100% cold ACN. After centrifugation at 12,000× g for 10 minutes, 550 μl of the supernatant was evaporated using SpeedVac. The dried sample was mixed with 20% methanol containing 0.1% formic acid, centrifuged at 12,000 x g for 10 minutes, and then a supernatant was obtained and injected into a C18 column for LC-MS/MS analysis.

1.5. Inhibition of UGT by lycolicidine in human liver microsomes

6 types of UGT-specific substrates (2.5 μM SN-38 for UGT1A1, 5 μM kenodioxycholic acid for UGT1A3, chenodeoxycholic acid for UGT1A4, 5 μM trifluoperazine for UGT1A4, 2.5 for UGT1A6 μM N -. it was tested acetyl serotonin (N -acetylserotonin), 1 μM with respect to the UGT1A9 mycophenolic acid (mycophenolic acid), inhibition of Ricoh receiver Dean for metabolism of 5 μM naloxone (naloxone) with respect to the six kinds of effect UGT2B7 To confirm the inhibitory effect of lycoricidine on the activity of UGT, each reaction was performed in pooled HLM (0.5 mg/ml) in 100 μl of the total culture volume 0.5 mg/ml alameticin 0.1 M potassium phosphate The culture mixture contained in the buffer solution (pH 7.4) was treated and stored on ice for 15 minutes to activate the enzyme.After pre-incubation for 5 minutes, UGT substrate cocktail and NGS were final concentrations of 0-25 μM ricolithine. And reacted for 60 minutes, the reaction was terminated by adding 100 μl of acetonitrile containing 0.1% formic acid, and 0.625 μM reserpine was added as an internal standard (IS). This was mixed, centrifuged at 13,000 x g for 10 minutes, and then injected into a C18 column by 10 µl to perform LC-MS/MS analysis.

1.6. M1 formation at various concentrations of human microsomes

0.5 mg/ml alameticin was treated with HLM (0.5 mg/ml or 1 mg/ml) and stored on ice for 15 minutes to activate the enzyme. Thereafter, 0.1 M of potassium phosphate buffer (pH 7.4) and 50 μM of lycoricidine were added to HLM and pre-incubated at 37° C. for 5 minutes. NGS solution was added to initiate the reaction, and the reaction was carried out for 60 minutes in the presence of 10 μM UDPGA, and then the reaction was terminated by adding several hundred μl of acetonitrile containing 0.1% formic acid. Vortex for 15 seconds, centrifuge for 10 minutes at 12,000 x g , then transfer the supernatant to a glass vial and perform LC-MS analysis.

1.7. LC-MS/MS analysis method

Nexera X2 Ultra High-Performance Liquid Chromatograph (Shimadzu, Kyoto, Japan) coupled with the Q Exactive Orbitrap Mass Spectrometer (Thermo Fisher Scientific Inc., MA, USA) was used to detect and reveal lyricidine and its metabolite structure. , Heated electrospray ionization (HESI) to which molecular ionization and negative ions were applied was monitored.

HESI conditions for identification of metabolites are as follows: sheath gas flow rate, 35 (arbitrary unit); Auxiliary gas flow rate, 10 (arbitrary unit); Spray voltage, 4 kV; And heater temperature, 350°C. Mass spectra were acquired at a resolution of 70,000 in the m/z range of 100 to 1000, and data-dependent MS ² spectra were acquired at a resolution of 35,000 using normalized collision energy at 23 and 28. The LC method consisted of 5% methanol with 0.1% formic acid (mobile phase A, mobile phase A) at a flow rate of 0.3 ml/min, and 95% methanol with 0.1% formic acid (mobile phase B, mobile phase B). Concentration gradient conditions include 0-0.5 min to 10% mobile phase B, 0.5-5.5 min to 10-90% mobile phase B, 5.5-8 min to 90% mobile phase B, 8-8.1 min to 90-10% Mobile phase B and 10% of mobile phase B at 8.1-10 min were used. The analyte was separated on a Halo C18 column (100 mm Х 2.1 mm, 2.7 μm , Advanced Materials Technology, Wilmington, DE, USA) while maintaining 40° C. After obtaining the MS data, it was processed using Xcalibur software 3.0 (Thermo Fisher Scientific, Inc.).

Quantification studies were performed using a TSQ Vantage Mass Spectrometer (Thermo Fisher Scientific Inc., MA, USA) comprising a HESI source linked to a Shimadzu HPLC (Shimadzu, Kyoto, Japan) system. The HPLC system consists of an LC-20AD pump, a SIL-20A autosampler and a CTO-20A column oven. Shim-pack GIS column (3 μM ODS, 150 mm×3 mm) (Shimadzu, Kyoto, Japan) was used for LC separation. The mass spectrometer was operated in positive ESI mode with nitrogen set at 10 and 35 psi, respectively, for the auxiliary gas and sheath gas pressures. The ESI spray voltage was adjusted to 4,000 V, the temperature of the evaporator was set to 300°C, and the temperature of the capillary tube was 350°C. Data were obtained using Xcalibur software.

In the metabolic stability study, solvent A (water with 0.1% formic acid) and solvent B (100% ACN with 0.1% formic acid) were mixed to apply a concentration gradient program to the mobile phase at a flow rate of 500 μl/min. Concentration gradients applied were: 5-5% B (0-0.5 min), 5-95% B (0.5-3.5 min), 95-95% B (3.5-5 min), 95-5% B ( 5-5.5 minutes) and 5-5% B (5.5-9 minutes). During the analysis, the column oven was maintained at 40°C. In the UGT enzyme inhibition assay, gradient elution was performed as follows: 0-0.5 min to 5% B, 0.5-4 min to 5-95% B, 4-6 min to 95% B, 6- B of 95-5% at 6.1 min, 5% B at 6.1-10 min.

2. Experimental results

2.1. Confirmation of glucuronation of lyricidine

The metabolic stability of ricolithidine shows that glucuronation of ricolithidine is the preferred metabolic result in Phase I metabolism in HLM (FIG. 2 ). As observed in Phase II metabolism, after 60 minutes of incubation, lyricidine levels decreased by about 7% and 59% at 10 μM and 50 μM, respectively (FIG. 2A). In particular, it was found that the level of lycoricidine decreased rapidly to 38% in 10 μM, 15 minute incubation. However, in the Phase I metabolic system, the stability of Lachoricidine did not decrease when UDPGA was not present (FIG. 2B ).

2.2. Structural analysis of the lyricidine metabolite M1

M1, one of the lycoricidine metabolites, was confirmed at a retention time of 6.1 minutes after culturing lycoricidine with HLM with UDPGA and NGS added (FIG. 3B). For the interpretation of the M1 structure, the MS ² spectra of lycoricidine and M1 were analyzed with a high resolution quadrupole-orbitrap mass spectrometer (Table 1).

Compounds Parent ions [M-H] ^- Elemental composition Error (ppm) Product ions (m/z) Elemental composition Error (ppm) CE Licoricidin





423.2181





C ₂₆ H ₃₁ O ₅





3.5





391.1919 C ₂₅ H ₂₇ O ₄ 3.9 28 245.1182 C ₁₅ H ₁₇ O ₃ 4.1 28 233.1179 C ₁₄ H ₁₇ O ₃ 3.1 28 219.1021 C ₁₃ H ₁₅ O ₃ 2.2 28 207.1019 C ₁₂ H ₁₅ O ₃ 1.6 28 203.1069 C ₁₃ H ₁₅ O ₂ 1.4 28 177.0909 C ₁₁ H ₁₃ O ₂ -0.5 28 M1
599.2504
C ₃₂ H ₃₉ O ₁₁
2.8
423.2178 C ₂₆ H ₃₁ O ₅ 2.8 23 175.0237 C ₆ H ₇ O ₆ -0.01 23

Precursor ion of Ricoh receiver Dean and M1 is in the voice mode [MH] ^- was detected in each _{423.2181 (C 26 H 31 O 5} ) and _{599.2254 (C 32 H 39 O 11} ). The M1 ion was as high as 176.0076 Da compared to lycoricidine, indicating the possibility of monoglucuronation in the hydroxyl group in the parent structure. The MS ² spectrum of lycoricidine is shown in Figure 4, which was obtained after high energy collision-induced dissociation in negative mode.

The M1 ion was observed at m/z 599.2254 corresponding to monoglucuronation, and the MS ² spectrum is shown in FIG. 5. M/z 423.2178 (C ₂₆ H ₃₁ O ₅ ) with an error of 2.8 ppm was detected after the loss of the glucuronide group (Table 1), and also the truncated glucuronic acid with an error of -0.01 ppm. m/z 175.0237 (C ₆ H ₇ O ₆ ). In the present invention, M1 is defined as mono-glucuronyl lyricidine.

2.3. Reactive phenotype of lyricidine glucuronation

It can be seen that the glucuronide binding of lyricidine is dependent on the culture time and the amount of microsome protein (FIG. 6A ). When lycoccidin was incubated with recombinant human UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7, it was confirmed that UGT1A9 produced M1 most remarkably, and UGT2B7 and UGT1A3 contributed somewhat to the formation of M1 ( 2B). No metabolites were observed in other UGT isozymes. The inventors observed that when incubated with human recombinant UGT1A9, the level of lycolicidine, the accepted UGT1A9 substrate, decreased rapidly compared to mycophenolic acid, which was stronger and more specific to UGT1A9 when lycoricidine was compared to mycophenolic acid. It indicates that it is a natural substrate (B in Fig. 6). In addition, lycoridine showed a somewhat inhibitory effect on UGT1A9-catalyzed mycophenolic acid glucuronation with an IC ₅₀ of 46.8±3.0 μM. In addition, lycoricidine showed a pronounced inhibitory effect on UGT1A3-catalyst kenodioxycholic glucuronation (IC ₅₀ = 24.7 ± 6.9) and UGT2B7-catalyzed naloxone glucuronation (IC ₅₀ = 28.6 ± 4.4 μM) ( Table 2).

Isoforms Substrates Cons (μM) IC ₅₀ UGT1A1 SN-38 2.5 - UGT1A3 Chenodeoxycholic acid 5 24.7 ± 6.9 UGT1A4 Trifluoprazine 5 - UGT1A6 N -Acetylserotonin 2.5 - UGT2B7 Naloxone 5 28.6 ± 4.4

From these results, it was confirmed that UGT1A9 is a major enzyme that produces monoglucuronide lyricidine. Lycoricidine showed higher metabolic rate with recombinant human UGT1A9 compared to mycophenolic acid in the presence of NADPH and UDPGA. From the selective metabolic activity of UGT1A9 to lycoricidine glucuronidation, the present inventors have confirmed that lyricidine can be used as a substrate for new UGT1A9 to determine human UGT1A9 activity. These results can provide useful information for studying metabolism of bioactive natural products.

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So far, the present invention has been focused on the preferred embodiments. Those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in terms of explanation, not limitation. The scope of the present invention is indicated by the following claims rather than the above detailed description, and it is interpreted that all modifications or variations derived from the meaning and scope of the claims and equivalent concepts are included in the scope of the present invention. Should be.

Claims (6)

Uridine 5`-diphosphoglucuronosyltransferase (UGT) activity evaluation composition containing lycoricidine.
The composition according to claim 1, wherein the uridine 5`-diphosphoglucuronosyltransferase is at least one selected from the group consisting of UGT1A9, UGT2B7 and UGT1A3.
Licoricedin, glucose-6-phosphate, β-NADP+ and glucose-6-phosphate dehydrogenase and UDP-glucuronic acid Uridine 5`-diphosphoglucuronosyltransferase activity evaluation kit comprising a.
a) contacting the composition of claim 1 with a new drug candidate;
b) incubating by adding glucose-6-phosphate, β-NADP+ and glucose-6-phosphate dehydrogenase to the result of step a);
c) culturing by adding UDP-glucuronic acid to the product of step b);
d) performing mass spectrometry of the culture result of step c); And
e) Method for evaluating the activity of uridine 5`-diphosphoglucuronosyltransferase of a new drug candidate substance comprising the step of confirming the presence or absence of monoglucuronide lyricidine from the results of the mass spectrometry.
The method of claim 4, wherein the uridine 5`-diphosphoglucuronosyltransferase is at least one selected from the group consisting of UGT1A9, UGT2B7 and UGT1A3.
The method according to claim 4, wherein the mass spectrometry is performed by LC-MS/MS.

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