Inhibitory Potential of Bilobetin Against CYP2J2 Activities in Human Liver Microsomes

( Zhexue Wu ),( Su-nyeong Jang ),( So-young Park ),( Nguyen Minh Phuc ),( Kwang-hyeon Liu ) 한국질량분석학회 2020 Mass spectrometry letters Vol.11 No.4

Cytochrome P450 2J2 (CYP2J2) is a member of the cytochrome P450 superfamily, and is known to be arachidonic acid epoxygenase that mediates the formation of four bioactive regioisomers of epoxyeicosatrienoic acids (EETs). CYP2J2 is also involved in the metabolism of drugs such as albendazole, astemizole, danazol, ebastine, and terfenadine. CYP2J2 is highly expressed in the heart and cancer tissues. In this study, the inhibitory potential of ten natural products against CYP2J2 activity was evaluated using human liver microsomes and tandem mass spectrometry. Among them, bilobetin, which is a kind of biflavonoid, exhibits a strong inhibitory effect against the CYP2J2-mediated astemizole O-demethylation (IC₅₀ = 0.73 μM) and terfenadine hydroxylation (IC₅₀ = 0.89 μM). This result suggests that bilobetin can be used as strong CYP2J2 inhibitor in drug metabolism study.

Structural identification of skin ceramides containing x-hydroxy acyl chains using mass spectrometry

Zhexue Wu,손종철,김종례,조윤희,유광현 대한약학회 2016 Archives of Pharmacal Research Vol.39 No.10

The stratum corneum (SC) acts as a barrier that protects organisms against the environment and from transepidermal water loss. It consists of corneocytes embedded in a matrix of lipid metabolites (ceramides, cholesterol, and free fatty acids). Of these lipids, ceramides are sphingolipids consisting of sphingoid bases, linked to fatty acyl chains. Typical fatty acid acyl chains are composed of a-hydroxy fatty acids (A), esterified x-hydroxy fatty acids (EO), non-hydroxy fatty acids (N), and x-hydroxy fatty acids (O). Of these, O-type ceramides are esterlinked via their x-hydroxyl group to proteins in thecornified envelope and can be released and extracted following mild alkaline hydrolysis. Tandem mass spectrometry (MS/MS) analysis of O-type ceramides using chipbased direct infusion nanoelectrospray-ion trap mass spectrometry generated the characteristic fragmentation pattern of both acyl and sphingoid units, suggesting that this method could be applied to the structural identification of O-type ceramides. Based on the MS/MS fragmentation patterns of O-type ceramides, comprehensive fragmentation schemes are proposed. In addition, we have also developed a method for identifying and profiling O-type ceramides in the mouse and guinea pig SC. This information may be used to identify O-type ceramides in the SC of animal skin.

Thelephoric acid의 CYP2J2 효소 활성 저해제 평가

오철학(Zhexue Wu),이보람(Boram Lee),송경식(Kyung-Sik Song),류광현(Kwang-Hyeon Liu) 한국생명과학회 2013 생명과학회지 Vol.23 No.9

CYP2J2 효소는 간외의 조직에 존재 하는 효소로써, 주로 심혈관계에 발현되어 있다. CYP2J2는 내인성 대사체 및 여러 치료 약물들의 대사에 중요한 작용을 하고 있다. 또한 CYP2J2는 인체의 종양조직이나 종양 세포주에서 과발현되어 있어, 종양 치료를 위한 새로운 표적이 되고 있다. 본 연구에서는 천연물 10종을 대상으로 시토크롬 2J2 동효소에 저해능을 가지는 화합물을 발굴하고자 하였다. 10종의 천연물 중 thelephoric acid는 CYP2J2에 의해 매개되는 에바스틴(IC50=5.32 μM), 아스테미졸(IC50=3.23 μM) 및 터페나딘(IC50=3.27 μM) 대사를 강력하게 저해하였다. 향후, 이 약물을 대상으로 한 항암 활성 평가가 필요할 것으로 판단된다. Cytochrome P450 2J2 (CYP2J2) is an enzyme mainly found in human extrahepatic tissues, with predominant expression in the cardiovascular system. CYP2J2 plays important roles in the metabolism of endogenous metabolites and therapeutic drugs, such as arachidonic acid, astemizole, ebastine, and terfenadine. CYP2J2 is also overexpressed in human cancer tissues and cancer cell lines and may represent a potential target for therapy of human cancers. In this study, 10 natural products obtained from plants and microorganisms were screened as potential CYP2J2 inhibitors. Among them, thelephoric acid showed strong inhibition of astemizole O-demethylation activity (IC50=3.23 μM) in a dose-dependent manner. Evaluation of the substrate dependency of the inhibitory activity of thelephoric acid showed that it strongly inhibited CYP2J2-mediated ebastine hydroxylation (IC50=5.32 μM) and terfenadine hydroxylation (IC50=3.27 μM) in a substrate nondependent manner. The present data suggest that this compound might be a potential candidate for further evaluation for anticancer activity.

분광광도계를 이용한 폴리에톡시레이티드 아스코르빈산 분석법 개발

오철학 ( Zhexue Wu ),류광현 ( Kwang-hyeon Liu ) 한국응용생명화학회(구 한국농화학회) 2016 Journal of Applied Biological Chemistry (J. Appl. Vol.59 No.4

본 연구에서는 3-ethyl ascorbic acid를 표준물질로 이용하여, 폴리에톡시레이티드 아스코르빈산의 정량법을 개발하였고, 확립된시험법으로 시중에서 돼지 및 닭의 면역력 증진과 열에 대한 스트레스 경감제로 유통되고 있는 Poustin-C 검체 내 폴리에톡시레이티드 아스코르빈산의 함량 분석을 실제로 분석하여 시험법의 유효성을 검증하였다. 개발한 분석법의 검증은 직선성, 정확성, 정밀성 및 안정성 시험 등을 통하여 평가하였다. 정밀도 시험은 일내 정밀성과 일간 정밀성으로 나누어 확인하였는데, 일내 및 일간 정밀성은 모두 3.4 % 이내로 매우 우수하였다. 그리고 직선성 실험에서는 상관계수(r²)가 0.998 이상으로 우수하였다. 실온에서 6시간까지는 안정하여 실험 도중 분해되지 않음을 확인하였다. 따라서 본 연구에서 개발된 시험법은 검체 내폴리에톡시레이티드 아스코르빈산의 함량을 분석하기 위한 공정 시험법으로 활용할 수 있을 것으로 사료된다. We developed a spectrophotometric assay method for polyethoxylated ascorbic acidusing 3-ethyl ascorbic acid as standard material. The analytical method was validated by linearity, accuracy, precision, and stability. The coefficient of variation of the precision of the assay was less than 3.4 %. The linearity of the calibration curves in the desired concentration range is good (r ² >0.998). 3-Ethyl ascorbic acid and polyethoxylated ascorbic acid were stable in stock solution at room temperature for up to at least 6 h. The developed assay could be used for the content analysis of polyethoxylated ascorbic acid in samples.

CYP2J2 and CYP2C19 Are the Major Enzymes Responsible for Metabolism of Albendazole and Fenbendazole in Human Liver Microsomes and Recombinant P450 Assay Systems

Wu, Zhexue,Lee, Doohyun,Joo, Jeongmin,Shin, Jung-Hoon,Kang, Wonku,Oh, Sangtaek,Lee, Do Yup,Lee, Su-Jun,Yea, Sung Su,Lee, Hye Suk,Lee, Taeho,Liu, Kwang-Hyeon American Society for Microbiology 2013 Antimicrobial agents and chemotherapy Vol.57 No.11

<P>Albendazole and fenbendazole are broad-spectrum anthelmintics that undergo extensive metabolism to form hydroxyl and sulfoxide metabolites. Although CYP3A and flavin-containing monooxygenase have been implicated in sulfoxide metabolite formation, the enzymes responsible for hydroxyl metabolite formation have not been identified. In this study, we used human liver microsomes and recombinant cytochrome P450s (P450s) to characterize the enzymes involved in the formation of hydroxyalbendazole and hydroxyfenbendazole from albendazole and fenbendazole, respectively. Of the 10 recombinant P450s, CYP2J2 and/or CYP2C19 was the predominant enzyme catalyzing the hydroxylation of albendazole and fenbendazole. Albendazole hydroxylation to hydroxyalbendazole is primarily mediated by CYP2J2 (0.34 μl/min/pmol P450, which is a rate 3.9- and 8.1-fold higher than the rates for CYP2C19 and CYP2E1, respectively), whereas CYP2C19 and CYP2J2 contributed to the formation of hydroxyfenbendazole from fenbendazole (2.68 and 1.94 μl/min/pmol P450 for CYP2C19 and CYP2J2, respectively, which are rates 11.7- and 8.4-fold higher than the rate for CYP2D6). Correlation analysis between the known P450 enzyme activities and the rate of hydroxyalbendazole and hydroxyfenbendazole formation in samples from 14 human liver microsomes showed that albendazole hydroxylation correlates with CYP2J2 activity and fenbendazole hydroxylation correlates with CYP2C19 and CYP2J2 activities. These findings were supported by a P450 isoform-selective inhibition study in human liver microsomes. In conclusion, our data for the first time suggest that albendazole hydroxylation is primarily catalyzed by CYP2J2, whereas fenbendazole hydroxylation is preferentially catalyzed by CYP2C19 and CYP2J2. The present data will be useful in understanding the pharmacokinetics and drug interactions of albendazole and fenbendazole <I>in vivo</I>.</P>

Lipidomic platform for structural identification of skin ceramides with α-hydroxyacyl chains

Wu, Zhexue,Shon, Jong Cheol,Lee, Doohyun,Park, Kab-Tae,Park, Chang Seo,Lee, Taeho,Lee, Hye Suk,Liu, Kwang-Hyeon Springer-Verlag 2016 Analytical and Bioanalytical Chemistry Vol.408 No.8

<P>Skin ceramides are sphingolipids consisting of sphingoid bases, which are linked to fatty acids via an amide bond. Typical fatty acid acyl chains are composed of alpha-hydroxy fatty acid (A), esterified omega-hydroxy fatty acid (EO), non-hydroxy fatty acid (N), and omega-hydroxy fatty acid (O). We recently established a lipidomic platform to identify skin ceramides with non-hydroxyacyl chains using tandem mass spectrometry. We expanded our study to establish a lipidomic platform to identify skin ceramides with alpha-hydroxyacyl chains. Tandem mass spectrometry analysis of A-type ceramides using chip-based direct infusion nanoelectrospray-mass spectrometry showed the characteristic fragmentation pattern of both acyl and sphingoid units, which can be applied for structural identification of ceramides. Based on the tandem mass spectrometry fragmentation patterns of A-type ceramides, comprehensive fragmentation schemes were proposed. Our results may be useful for identifying A-type ceramides in the stratum corneum of human skin.</P>

<i>In vitro</i>characterization of 4′-(<i>p</i>-toluenesulfonylamide)-4-hydroxychalcone using human liver microsomes and recombinant cytochrome P450s

Lee, Boram,Wu, Zhexue,Lee, Taeho,Tan, Xue Fei,Park, Ki Hun,Liu, Kwang-Hyeon Informa UK (Informa Healthcare) 2016 Xenobiotica Vol.46 No.4

Danazol Inhibits Cytochrome P450 2J2 Activity in a Substrate-independent Manner

Lee, Eunyoung,Wu, Zhexue,Shon, Jong Cheol,Liu, Kwang-Hyeon American Society for Pharmacology and Experimental 2015 Drug metabolism and disposition: the biological fa Vol.43 No.8

<P>Cytochrome P450 2J2 (CYP2J2) is an enzyme responsible for the metabolism of endogenous substrates including arachidonic acid, as well as therapeutic drugs such as albendazole, astemizole, ebastine, and terfenadine. Selective inhibitors of CYP2J2 are essential for P450 reaction phenotyping studies. To find representative CYP2J2 index inhibitors, we evaluated the inhibitory potential of danazol, hydroxyebastine, telmisartan, and terfenadone against CYP2J2 activity for four representative CYP2J2 substrates (albendazole, astemizole, ebastine, and terfenadine) using recombinant CYP2J2. Of these four CYP2J2 inhibitors, danazol strongly inhibited CYP2J2-mediated albendazole, astemizole, ebastine, and terfenadine metabolism in a substrate-independent manner, with IC<SUB>50</SUB> values of 0.05, 0.07, 0.18, and 0.34 <I>μ</I>M, respectively. Danazol noncompetitively inhibited CYP2J2-mediated astemizole <I>O</I>-demethylation activities with a <I>K</I><SUB>i</SUB> value of 0.06 <I>μ</I>M. Terfenadone strongly inhibited CYP2J2-mediated albendazole, astemizole, and terfenadine metabolism (IC<SUB>50</SUB> < 0.21 <I>μ</I>M), whereas it showed weak inhibition against CYP2J2-catalyzed ebastine hydroxylase activity (IC<SUB>50</SUB> = 6.04 <I>μ</I>M). Telmisartan had no inhibitory effect on CYP2J2-mediated ebastine and terfenadine hydroxylation (IC<SUB>50</SUB> > 20 <I>μ</I>M). Taken together, these data suggest that danazol may be used as a CYP2J2 index inhibitor in reaction phenotyping studies.</P>

LKY-047: First Selective Inhibitor of Cytochrome P450 2J2

Phuc, Nguyen Minh,Wu, Zhexue,O, Yuseok,Lee, Jee-Hyun,Oh, Sangtaek,Song, Gyu-Yong,Liu, Kwang-Hyeon American Society for Pharmacology and Experimental 2017 Drug metabolism and disposition Vol.45 No.7

<P>Highly selective cytochrome P450 CYP2J2 (CYP2J2) inhibitors suitable for reaction phenotyping are currently not available. (7S)-(+)-(4-Nitro-phenyl)-acrylic acid, 8,8-dimethyl-2-oxo-6,7-dihydro2H, 8H-pyrano[ 3,2-g] chromen-7-yl-ester (LKY-047), a decursin derivative, was synthesized, and its inhibitor potencies toward CYP2J2 as well as other cytochrome P450 (P450) enzymes in human liver microsomes (HLM) were evaluated. LKY-047 was demonstrated to be a strong competitive inhibitor of CYP2J2-mediated astemizole O-demethylase and terfenadine hydroxylase activity, with K-i values of 0.96 and 2.61 mu M, respectively. It also acted as an uncompetitive inhibitor of CYP2J2-mediated ebastine hydroxylation with a K-i value of 3.61 mu M. Preincubation of LKY-047 with HLMs and NADPH did not alter inhibition potency, indicating that it is not a mechanism-based inhibitor. LKY-047 was found to be a selective CYP2J2 inhibitor with no inhibitory effect on other human P450s, such as CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A (IC50 > 50 mu M). These in vitro data support the use of LKY-047 as a selective CYP2J2 inhibitor with potential application in the identification of P450 isoforms responsible for drug metabolism in reaction phenotyping assays.</P>

Terfenadone is a strong inhibitor of CYP2J2 present in the human liver and intestinal microsomes

Lee, Eunyoung,Kim, Ju-Hyun,Shon, Jong Cheol,Wu, Zhexue,Kim, Hyun Ji,Gim, Minsik,Lee, Taeho,Liu, Kwang-Hyeon Elsevier 2018 Drug metabolism and pharmacokinetics Vol.33 No.3