A composition for inhibiting the activity of uridine diphosphate glucuronosyltransferase 1A9 and use thereof

Abstract

The application discloses a kind of compositions for inhibiting uridine diphosphate glucuronosyltransferase 1A9 activity and application thereof, belong to medical technology field.The composition includes two kinds or more in alpha-antiarol, beta-antiarol, gamma-antiarol, garcinia C, garcinia D, 8-deoxy-garcinol, garcinol and 3-isoantiarol.The application verifies by experiment that the extract of fruit mountain bamboo has higher affinity and stronger inhibitory activity to UGT1A9 in alpha-antiarol, beta-antiarol, gamma-antiarol, garcinia C, garcinia D, 8-deoxy-garcinol, garcinol and 3-isoantiarol, and the components have higher biological activity and safety, can be used as the selective inhibitor of UGT1A9, to inhibit the metabolism of clinical immunosuppressive drug mycophenolic acid, increase mycophenolic acid exposure, prolong the half-life of mycophenolic acid, improve mycophenolic acid efficacy.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a composition that inhibits the activity of uridine diphosphate glucuronide transferase 1A9 and its application. Background Technology

[0002] Urate diphosphate glucuronyl transferase 1A9 (UGT1A9) is a particularly important member of the UGT superfamily, highly expressed in metabolically active organs such as the liver and kidneys. UGT1A9 plays a crucial role in the inactivation / detoxification of many exogenous drugs (such as mycophenolate mofetil, propofol, and dapagliflozin) and is responsible for maintaining the homeostasis of endogenous signaling molecules (such as thyroid hormones, fatty acids, and estrogen). Furthermore, the expression and function of UGT1A9 are regulated by internal and external factors such as age, sex, inhibition, induction, and genetic determinants, leading to changes in drug clearance, efficacy / adverse reactions, and disease susceptibility. Therefore, co-administering UGT1A9 inhibitors to modulate the catalytic activity of the UGT1A9 enzyme is a potentially effective and practical strategy for improving the exposure and efficacy of drugs primarily metabolized through glucuronidation.

[0003] Mycophenolate mofetil is a commonly used immunosuppressant in clinical practice, primarily used to prevent and treat acute rejection after organ transplantation, and autoimmune diseases such as systemic lupus erythematosus, psoriasis, and rheumatoid arthritis. The high clinical dosage and long treatment period of mycophenolate mofetil lead to severe gastrointestinal toxicity. In vivo, mycophenolate mofetil is hydrolyzed by esterases to produce the active metabolite mycophenolic acid. Mycophenolic acid is then metabolized in vivo through a UGT1A9-mediated glucuronidation reaction to mycophenolic acid glucuronide. Approximately 87% of mycophenolate mofetil is excreted in the urine as mycophenolic acid glucuronide. The extensive glucuronidation metabolism of mycophenolic acid in the body significantly reduces its in vivo exposure, activity, and therapeutic efficacy, requiring higher doses to achieve the optimal therapeutic effect of mycophenolate mofetil. Therefore, developing a safe and effective UGT1A9 inhibitor to regulate the metabolism of mycophenolate mofetil, reduce the dosage of mycophenolate mofetil, increase its systemic exposure, prolong its half-life, and restore the sensitivity of immune cells may be a key step in improving the efficacy of mycophenolate mofetil and reducing its gastrointestinal toxicity.

[0004] Mangosteen, also known as mangosteen fruit, is the fruit of the evergreen tree *Garcinia mangosteen*, belonging to the genus *Garcinia* of the family Clusiaceae. Mangosteen fruit contains vitamin B, various amino acids, protein, and abundant minerals, possessing high nutritional value and earning the title "Queen of Tropical Fruits." The mangosteen husk is a traditional medicinal material in countries such as Thailand, Myanmar, and India, widely used to treat inflammation, wounds, and skin and injury infections. Mangosteen contains various secondary metabolites, which are increasingly attracting attention from the medical community. However, the inhibitory effect of mangosteen and its chemical components on UGT1A9 has not been reported, nor has there been any report on using mangosteen extracts and their UGT1A9 active inhibitors to inhibit mycophenolic acid metabolism, increase mycophenolic acid exposure, prolong mycophenolic acid half-life, or improve mycophenolic acid efficacy. Therefore, a comprehensive and systematic study is urgently needed on the potential inhibitory effect of mangosteen and its active components on UGT1A9, and the feasibility of using mangosteen / active components to inhibit mycophenolic acid metabolism, increase mycophenolic acid exposure, prolong mycophenolic acid half-life, and improve mycophenolic acid efficacy. Summary of the Invention

[0005] The purpose of this invention is to provide a composition and its application for inhibiting the activity of uridine diphosphate glucuronide transferase 1A9, in order to solve the problems existing in the prior art. This invention extracts active ingredients from the traditional Chinese medicine mangosteen, which are safe and non-toxic, and can be used to inhibit the metabolism of the clinical immunosuppressive drug mycophenolic acid, increase the exposure of mycophenolic acid, prolong the half-life of mycophenolic acid, and improve the efficacy of mycophenolic acid.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides the application of mangosteen extract in the preparation of uridine diphosphate glucuronyl transferase 1A9 inhibitor, wherein the mangosteen extract comprises one or more of α-dextrin, β-dextrin, γ-dextrin, gamboge C, gamboge D, 8-deoxygambogein, gambogein and 3-isodextrin.

[0008] Furthermore, the dosage forms of the uridine diphosphate glucuronyl transferase 1A9 inhibitor include tablets, pills, granules, and mixtures.

[0009] The present invention also provides the use of mangosteen extract in the preparation of a drug that inhibits mycophenolic acid metabolism, wherein the mangosteen extract comprises one or more of α-dextrin, β-dextrin, γ-dextrin, gamboge C, gamboge D, 8-deoxygambogein, gambogein and 3-isodextrin.

[0010] Furthermore, the inhibition of mycophenolic acid metabolism includes prolonging the half-life of mycophenolic acid and increasing mycophenolic acid exposure.

[0011] Furthermore, the dosage forms of the drug include tablets, pills, granules, and mixtures.

[0012] The present invention also provides the use of mangosteen extract in the preparation of a drug that enhances the immunosuppressive activity of mycophenolic acid, wherein the mangosteen extract comprises one or more of α-dextrin, β-dextrin, γ-dextrin, gamboge C, gamboge D, 8-deoxygambogein, gambogein and 3-isodextrin.

[0013] Furthermore, the mangosteen extract enhances the immunosuppressive activity of mycophenolic acid by inhibiting the activity of uridine diphosphate glucuronyltransferase 1A9 and thus inhibiting mycophenolic acid metabolism.

[0014] Furthermore, the dosage forms of the drug include tablets, pills, granules, and mixtures.

[0015] The present invention also provides a composition for inhibiting the activity of uridine diphosphate glucuronyl transferase 1A9, the composition comprising two or more of α-dextrin, β-dextrin, γ-dextrin, gamboge C, gamboge D, 8-deoxygambogein, gambogein and 3-isodextrin.

[0016] The present invention discloses the following technical effects:

[0017] This invention experimentally verified that α-dextrin, β-dextrin, γ-dextrin, gamboge C, gamboge D, 8-deoxygambogein, gambogein and 3-isodextrin in mangosteen extract have high affinity and strong inhibitory activity for UGT1A9. At the same time, these components have high biological activity and safety and can be used as selective inhibitors of UGT1A9.

[0018] This invention, through in vitro activity assays, revealed that the above-mentioned inhibitor inhibits the UGT1A9-mediated mycophenolic acid glucuronidation reaction with an IC50 value. 50 and K i With values ​​between 55 and 971 nM and 87.7 and 418 nM respectively, it can be used to inhibit in vitro metabolic reactions of endogenous or exogenous substances mediated by uridine diphosphate glucuronyl transferase 1A9.

[0019] This invention extracts active ingredients from fruits, which are safe and non-toxic. These ingredients can be used to inhibit the metabolism of mycophenolic acid, a clinical immunosuppressive drug, increase mycophenolic acid exposure, prolong the half-life of mycophenolic acid, and improve the efficacy of mycophenolic acid. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The inhibitory effect of one hundred kinds of Chinese herbal extracts on UGT1A9 activity;

[0022] Figure 2 The inhibition curve of mangosteen extract on UGT1A9;

[0023] Figure 3 The chemical fingerprint of mangosteen extract (A), the spectrum of the inhibitory effect of mangosteen extract fraction on UGT1A9 (B), and the chemical structural formula of the active substance after identification (C) are shown.

[0024] Figure 4 Figure showing the inhibition of mycophenolic acid metabolism by a series of compounds from mangosteen extract at the HepaRG cell level;

[0025] Figure 5 Cytotoxicity of mangosteen monomer components combined with mycophenolic acid at the HepaRG cell level;

[0026] Figure 6 The effect of a series of compounds from mangosteen extract on the concentrations of mycophenolic acid and mycophenolic acid glucuronide in HepaRG cell supernatant;

[0027] Figure 7 The metabolic pattern of mycophenolic acid during the combined use of mycophenolic acid and α-dextrin;

[0028] Figure 8 To enhance the inhibitory effect of mycophenolic acid on lipopolysaccharide-induced proliferation of primary mouse spleen B lymphocytes by α-dextrin. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0033] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0034] The inventors conducted research on the inhibitory effects of over one hundred kinds of Chinese herbal extracts and their main chemical components on UGT1A9. Figure 1 This study aimed to identify safe and effective UGT1A9 inhibitors from common Chinese herbal medicines to inhibit mycophenolic acid metabolism, increase mycophenolic acid exposure, prolong its half-life, and improve its efficacy. The study found that mangosteen extract and its monomeric components can effectively inhibit UGT1A9 activity. The active components of mangosteen extract are flavonoids, including α-mangostin, β-mangostin, γ-mangostin, garcinone C, garcinone D, 8-desoxygartanin, gartanin, and 3-isomangostin, with the following structural formulas:

[0035]

[0036] In the following examples, the above-mentioned mangosteen extract is collectively referred to as mangosteen extract D6. The extraction method is as follows: 50g of mangosteen peel is added to 500mL of 95% ethanol, sealed with plastic wrap, and soaked at room temperature for 4 hours. After the herb has been completely soaked, it is sonicated for 2 hours, filtered to remove the residue, and then rotary evaporated in a large bottle until it becomes viscous or dry. The residue is then scraped off and transferred to a white plastic bottle. After cooling to room temperature, the bottle cap is tightened and the bottle is labeled and stored at -20℃.

[0037] Example 1: Determination of the inhibitory activity of mangosteen extract on human UGT1A9

[0038] Using DDAO glucuronidation metabolism as a probe reaction and employing an in vitro UGTs enzyme incubation system, a multifunctional ELISA plate assay was used for high-throughput screening of medicinal materials with inhibitory activity against human UGT1A9. The specific experimental procedure is as follows:

[0039] (1) The in vitro UGT metabolism reaction system included Tris-HCl buffer (50mM) at pH 7.4, MgCl2 solution (5mM), mixed human liver microsomes (0.1mg / mL), DDAO final concentration of 3.0μM, mangosteen extract D6 (10μg / mL), and pre-incubated with shaking at 37℃ for 5min;

[0040] (2) Initiate the reaction by adding the initiating factor UDPGA (final concentration 2.5 mM) to the reaction system;

[0041] (3) After 30 min, add 200 μL of ice-cold acetonitrile, shake vigorously, and then terminate the reaction;

[0042] (4) After centrifugation at 4℃ and 20,000×g for 10 min, the supernatant was collected and fluorescence detection was performed (Ex=465nm, Em=608nm);

[0043] The results are as follows Figure 2 As shown, mangosteen extract D6 exhibited strong inhibitory activity against UGT1A9, with a residual activity of 7% for UGT1A9 in this reaction system, IC50... 50 The value was 0.26 μg / mL.

[0044] Example 2: Tracking of UGT1A9 Inhibitors in Mangosteen under Activity-Directed Pathway

[0045] A fingerprint chromatogram of mangosteen extract was established using high-performance liquid chromatography (HPLC). The eluents were collected, and the inhibitory activity of all eluents against UGT1A9 was determined using DDAO glucuronidation metabolism as a probe reaction. The fingerprint chromatograms and inhibition chromatograms were compared, and the main monomeric components of mangosteen that inhibit UGT1A9 were identified by comparing retention time, UV spectroscopy, and high-resolution mass spectrometry with those of standards. The specific experimental procedure is as follows:

[0046] (1) Fingerprint establishment: A fingerprint of mangosteen extract was established using Waters high-performance liquid chromatography (HPLC) with an Acquity UPLC HSS T3 C18 (2.1 × 100 mm, 1.8 μm) column. The mobile phase consisted of acetonitrile (A) and water (B), with a flow rate of 0.8 mL / min and a detection wavelength of 285 nm. The eluent was collected directly into a black 96-well plate every 60 s, vacuum dried, and used for UGT1A9 inhibition experiments.

[0047] (2) Establishment of inhibition spectrum: The inhibitory effect of monomeric compounds in mangosteen on UGT1A9 activity was evaluated using DDAO, a UGT1A9-specific fluorescent probe substrate. The incubation system contained HLM (0.1 mg / mL), 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 3.0 μM DDAO, and the chromatographic elution fraction of mangosteen, and was pre-incubated at 37 °C for 5 min. 10 μL of UDPGA was added to the reaction system to initiate the reaction. After 30 min, 200 μL of ice-cold acetonitrile was added, and the reaction was terminated after vigorous shaking. After centrifugation at 20,000 × g for 10 min at 4 °C, the supernatant was collected for fluorescence detection (Ex = 465 nm, Em = 608 nm).

[0048] The results are as follows Figure 3 As shown, eight components in the mangosteen extract exhibited strong inhibitory effects on UGT1A9. By comparing the retention time, UV spectroscopy, and high-resolution mass spectrometry with the standard, they were identified in sequence as α-dextrin, β-dextrin, γ-dextrin, gamboge C, gamboge D, 8-deoxygambogein, gambogein, and 3-isodextrin.

[0049] Example 3: In vitro inhibition assay to quantitatively evaluate the inhibitory ability of mangosteen series compounds on UGT1A9.

[0050] Using mycophenolic acid, propofol, and DDAO glucuronidation metabolism as probe reactions, and employing an in vitro UGTs enzyme incubation system, a multifunctional microplate assay was used to evaluate the IC50 of different concentrations of mangosteen extract monomers (α-dextrin, β-dextrin, γ-dextrin, gamboge C, gamboge D, 8-deoxygambogein, gambogein, and 3-isodextrin) on UGT1A9 inhibition in high throughput. 50 The Ki value and the specific experimental procedure are as follows:

[0051] (1) The in vitro UGT metabolism reaction system included Tris-HCl buffer (50mM) at pH 7.4, MgCl2 solution (5mM), mixed human liver microsomes (0.1mg / mL), mycophenolic acid / propofol / DDAO final concentrations of 30 / 100 / 1.0μM, and different concentrations of mangosteen extract monomer components, and was pre-incubated at 37℃ with shaking for 3min;

[0052] (2) Initiate the reaction by adding the initiating factor UDPGA (final concentration 2.5 mM) to the reaction system;

[0053] (3) After 30 min, add 200 μL of ice-cold acetonitrile, shake vigorously, and then terminate the reaction;

[0054] (4) After centrifugation at 4℃ and 20,000×g for 20 min, the supernatant was collected for fluorescence detection and mass spectrometry detection. The maximum excitation and emission wavelengths of DDAO-glucuronide were 465nm and 608nm, respectively. Mycophenolic glucuronide was detected in negative ion mode, with a mother ion of 495.0 and a daughter ion of 319.2. Propofol glucuronide was detected in negative ion mode, with a mother ion of 353.0 and a daughter ion of 177.0.

[0055] The results are shown in Table 1. All eight monomeric components of mangosteen exhibited inhibitory activity against UGT1A9-mediated mycophenolic acid and DDAO. α-Dystocin, β-Dystocin, γ-Dystocin, Gascin C, Gascin D, 3-Isodystocin, Garcinin, and 8-Deoxygarcinin showed inhibitory effects on the mycophenolic acid-O-glucuronidation IC50 of UGT1A9 inhibition. 50 The values ​​were 69.5, 345, 592, 86.5, 55, 971, 282, and 437 nM, respectively, and the Ki values ​​were 89.3, 392, 197, 87.7, 102.5, 394, 237.4, and 418 nM, respectively. Table 1 shows the IC50 values ​​of mangosteen monomer components inhibiting UGT1A9-mediated DDAO-O-glucuronidation and mycophenolic acid-O-glucuronidation metabolism. 50 and Ki value

[0056]

[0057]

[0058] Example 4: HepaRG cell level determination of the effects of mangosteen series compounds on mycophenolic acid metabolism

[0059] UGT1A9-overexpressing HepRG cells were established. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) was used to determine the content of mycophenolic acid metabolite (mycophenolic acid glucuronide) in the HepRG cell supernatant at different time points, investigating the effect of mangosteen monomer components on mycophenolic acid metabolism. The specific operational procedure is as follows:

[0060] (1) Establishment of the HepaRG cell-level mycophenolic acid metabolism system: HepaRG cells were cultured in a cell culture incubator (37℃, 5% CO2, 95% humidity) in 1640 medium containing 10% fetal bovine serum and 1% antibody (100 units / mL penicillin, 100 μg / mL streptomycin). The cells were seeded in 24-well cell culture plates and cultured for 1-2 days. Mycophenolic acid (5 μM) and different concentrations (0-20 μM) of mangosteen extract monomers were added and incubated for 12 h. The cell supernatant was then collected, centrifuged at high speed for 20 min, and the supernatant was collected for mass spectrometry analysis.

[0061] (2) HepaRG cells were seeded in 96-well cell culture plates, mycophenolic acid (5 μM) and different concentrations of mangosteen extract monomer components (0-20 μM) were added, and after incubation for 12 h, the culture medium was aspirated, 100 μL of CCK-8 solution was added to each well, and the plates were placed in an incubator in the dark for 50 min. The OD value at 450 nm was detected by an enzyme-linked immunosorbent assay reader.

[0062] (3) HepaRG cells were seeded in 24-well cell culture plates, mycophenolic acid (5 μM) and different mangosteen extract monomer components (20 μM) were added, and after co-incubation for 12 h, the cell supernatant was taken, centrifuged at high speed for 20 min, and the supernatant was taken for mass spectrometry to determine the changes in mycophenolic acid and mycophenolic acid glucuronide content.

[0063] (4) HepaRG cells were seeded in 24-well cell culture plates, and mycophenolic acid (5 μM) and α-dextrin (20 μM) were added. After incubation for 0, 1, 2, 3, 4, 5, 8 and 11 h, the cell supernatant was collected, centrifuged at high speed for 20 min, and the supernatant was collected for mass spectrometry to determine the metabolic pattern of mycophenolic acid and the production pattern of mycophenolic acid glucuronide.

[0064] The results are as follows Figure 4-7 As shown in Table 2, α-dextrin, β-dextrin, γ-dextrin, galacin C, galacin D, 3-isodextrin, garcinin, and 8-deoxygarcinin inhibited mycophenolic acid metabolism at the HepaRG live cell level. Table 2 shows the IC50 values. 50 The concentrations were 24.3, 97, 135.5, 90.5, 8.5, 680.6, 403.2, and 1115 nM, respectively. In UGT1A9-overexpressing HepaRG cells, all eight monomeric components of mangosteen significantly inhibited the metabolism of mycophenolic acid, increasing its concentration in the cell supernatant by approximately 12-fold. Simultaneously, α-dextrin prolonged the metabolic half-life of mycophenolic acid in UGT1A9-overexpressing HepaRG cells from 2.35 h to 17.24 h.

[0065] Table 2. Inhibition of mycophenolic acid metabolism IC50 by a series of compounds from mangosteen extract at the HepaRG cell level 50 numerical values

[0066]

[0067]

[0068] Example 5: Effect of α-Dextrin on the immunosuppressive activity of mycophenolic acid at the level of primary spleen cells in mice.

[0069] Primary spleen cells were isolated from female Kunming rats. Splenic B lymphocytes were induced to proliferate using lipopolysaccharide (LPS). Cell viability was determined using the CCK-8 assay. The effect of α-dextrin on the immunosuppressive activity of mycophenolic acid was investigated. The specific procedures are as follows:

[0070] (1) Establishment of the experimental system for proliferation of mouse spleen lymphocytes: Spleen tissue was collected from mice, and mouse spleen lymphocytes were obtained by centrifugation at 800×g using a mouse spleen lymphocyte isolation kit. The isolated mouse spleen lymphocytes were then seeded into 96-well plates with a cell count of 3×10⁶ cells / well. 5 In each well, except for the blank group, LPS was added to a final concentration of 8 μg / mL.

[0071] (2) Effect of α-dextrin on the immunosuppressive activity of mycophenolic acid: Different final concentrations of α-dextrin (1, 2 and 5 μM) and mycophenolic acid (0.01 μM) were added to mouse spleen lymphocytes that had been treated with LPS. The cell culture conditions were 37℃, 5% CO2, 95% humidity, and cultured for 48 h. 20 μL of CCK-8 solution was added and the cells were placed in an incubator in the dark for 5 h. The OD value at 450 nm was detected by microplate reader.

[0072] The results are as follows Figure 8 As shown, α-dextrin not only dose-dependently inhibited the proliferation of primary mouse spleen B lymphocytes at the level of mouse spleen lymphocytes, but also increased the inhibitory effect of mycophenolic acid on lipopolysaccharide-induced proliferation of primary mouse spleen B lymphocytes.

[0073] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of an active ingredient from mangosteen extract in the preparation of uridine diphosphate glucuronyl transferase 1A9 inhibitor, characterized in that, The active ingredient is Gashengwo D.

2. The application according to claim 1, characterized in that, The dosage forms of the uridine diphosphate glucuronyl transferase 1A9 inhibitor include tablets, pills, granules, and mixtures.

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