인체 간 조직의 Cytochrome P450 3A4의 활성에 대한 Troleandomycin의 작용기전

김복량,오현숙,김혜정 한국독성학회 1995 Toxicological Research Vol.11 No.2

Incubation of aflatoxin $B_1$ $(AFB_1)$ with microsomes isolated from human liver number 110 yielded two metabolite peaks which were aflatoxin $Q_1$ $(AFQ_1)$ and $(AFB_1)$-exo-8, 9-epoxide (exo-epoxide) in high performance liquid chromatography. Production ratio of $AFQ_1$ to exo-epoxide was 2.43$\pm $0.04. Metabolism of $(AFB_1)$ to $(AFQ_1)$ and exo-epoxide was inhibited by troleandomycin in a same degree although troleandomycin was not activated as a mechanism-based inhibitor. The inhibitory effect was dependent upon either the incubation time with $(AFB_1)$ or the preincubation time before the addition of $(AFB_1)$. Incubation of troleandomycin and NADPH by the microsomes resulted in the formation of a cytochrome P 450 (P450)-metabollc intermediate (MI) complex and the level was approximately 80% of total P450 3A4 in the microsomes. This figure was similar to that of the inhibitory effect of troleandomycin on $AFB_1$ metabolism. Glutathione which was reported that it prevented the formation of MI complex in rat liver microsomes did not inhibit the formation of MI complex in human liver microsomes. These results suggested that the inhibitory effect of troleandomycin on $AFB_1$ metabolism is due to the formation of MI complex with P450 3A4. And the reaction mechanism of troleandomycin by human liver microsomes might be dlfferent from that one by rat liver microsomes.

In Vitro Metabolism of a New Neuroprotective Agent, KR-31543 in the Human Liver Microsomes : Identification of Human Cytochrome P450

Ji, Hye-Young,Lee, Seung-Seok,Yoo, Sung-Eun,Kim, Hosoon,Lee, Dong-Ha,Lim, Hong,Lee, Hye-Suk The Pharmaceutical Society of Korea 2004 Archives of Pharmacal Research Vol.27 No.2

KR-31543, (2S,3R,4S)-6-amino-4-[N-(4-chlorophenyl)-N-(2 -methyl-2H-tetrazol-5-ylmethyl) amino]-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-2H-1-benzopyran, is a new neuroprotective agent for preventing ischemia-reperfusion damage. This study was performed to identify the metabolic pathway of KR-31543 in human liver microsomes and to characterize cytochrome P450 (CYP) enzymes that are involved in the metabolism of KR-31543. Human liver microsomal incubation of KR-31543 in the presence of NADPH resulted in the formation of two metabolites, M1 and M2. M1 was identified as N-(4-chlorophenyl)-N-(2-methyl-2H-tetrazol-5-ylmethyl)amine on the basis of LC/MS/MS analysis with a synthesized authentic standard, and M2 was suggested to be hydroxy-KR-31543. Correlation analysis between the known CYP enzyme activities and the rates of the formation of M 1 and M2 in the 12 human liver microsomes have showed significant correlations with testosterone 6$\beta$-hydroxylase activity (a marker of CYP3A4). Ketoconazole, a selective inhibitor of CYP3A4, and anti-CYP3A4 monoclonal antibodies potently inhibited both N-hydrolysis and hydroxylation of KR-31543 in human liver microsomes. These results provide evidence that CYP3A4 is the major isozyme responsible for the metabolism of KR-31543 to M1 and M2.

Human Liver Microsome을 이용한 수종 이기약의 간대사효소에 미치는 영향

김현호,신용철,고성규,Kim, Hyun-Ho,Shin, Yong-Cheol,Ko, Seong-Gyu 대한예방한의학회 2008 대한예방한의학회지 Vol.12 No.2

In this study, we experimented the influence of three herbal medicines, which are Saussurea lappa Clarke, Poncirus trifoliata Rafin, Citrus aurantium Linne, which are called 'Yigiyak(理氣藥)' on drug metabolizing enzyme cytochrome P450 3A4 in Human Liver Microsome. Above all, the reason for this study is that herbal medicines can be assumed that herbs might have interactions with drugs, other herbs, alcohol and chemicals whether those are much better synergy effects than expected effects when the medicine was treated alone or not. As a result, we showed that all of five traditional herbal medicines had no CYP 3A4 inhibition effect on 10, 20, 30, 40, $50{\mu}g/m{\ell}$ doses in Human Liver Microsome even Saussurea lappa Clarke showed a little inhibition as about 93% and 79% inhibition rate of control. However, this result are mostly not enough to prove that SLC has a CYP 3A4 inhibition effect. Moreover, it is not that those rates showed that those herbal medicines have CYP 3A4 induction effect. In conclusion, the result could support that those herbal medicines are more safe than chemical drugs even if this is the basic step to prove that result. Therefore, more specific studies to support this result, which are Kinetic study, cell and animal study then finally until clinical research, are required.

Biotransformation and molecular docking of cyazofamid by human liver microsomes and cDNA-expressed human recombinant P450s

Lee, H.,Kim, J.H.,Kim, E.,Shin, Y.,Lee, J.H.,Jung, H.,Lim, Y.,Lee, H.S.,Kim, J.H. The Korean Society for Applied Biological Chemistr 2016 Applied Biological Chemistry (Appl Biol Chem) Vol.59 No.4

The purpose of this study was to understand the formation of metabolites from the metabolic reaction of cyazofamid with human liver microsomes. Human liver microsomal incubation of cyazofamid in the presence of NADPH produced one metabolite, 4-chloro-2-cyano-5-(4-(hydroxymethyl)phenyl)N,N-dimethyl-1H-imidazole-1-sul fonamide (CCHS). An incubation study using cDNA-expressed human recombinant P450s (rCYPs) demonstrated that cyazofamid-derived CCHS is mediated by CYP2B6, 2C9, and 2C19 at different reaction rates. The crystal structure of cyazofamid was obtained using single-crystal X-ray diffraction. According to a molecular modeling study of the crystal structure of cyazofamid with the rCYPs 2B6, 2C9, 2C19, and 3A4, the metabolic reactivities (2B6 > 2C19 > 2C9) were well-correlated to the distances between heme irons of CYPs and 4-methylphenyl group of cyazofamid.

Biotransformation and molecular docking of cyazofamid by human liver microsomes and cDNA-expressed human recombinant P450s

H. Lee,J.H.Kim,E. Kim,Y. Shin,J. H. Lee,H. Jung,Y. Lim,이혜숙,김정한 한국응용생명화학회 2016 Applied Biological Chemistry (Appl Biol Chem) Vol.59 No.4

The purpose of this study was to understand the formation of metabolites from the metabolic reaction of cyazofamid with human liver microsomes. Human liver microsomal incubation of cyazofamid in the presence of NADPH produced one metabolite, 4-chloro-2-cyano-5-(4-(hydroxymethyl)phenyl)N,N-dimethyl-1H-imidazole-1-sulfonamide (CCHS). An incubation study using cDNA-expressed human recombinant P450s (rCYPs) demonstrated that cyazofamid-derived CCHS is mediated by CYP2B6, 2C9, and 2C19 at different reaction rates. The crystal structure of cyazofamid was obtained using single-crystal X-ray diffraction. According to a molecular modeling study of the crystal structure of cyazofamid with the rCYPs 2B6, 2C9, 2C19, and 3A4, the metabolic reactivities (2B6 > 2C19 > 2C9) were well-correlated to the distances between heme irons of CYPs and 4-methylphenyl group of cyazofamid.

감초 물 추출물 및 Glycyrrhizin이 인체 간 Microsome에서 Cytochrome P450 약물대사효소에 미치는 영향

박종훈,박지영,주영승,Park Jong-Hoon,Park Ji-Young,Ju Young-Sung 대한예방한의학회 2003 대한예방한의학회지 Vol.7 No.2

Objective : The aim of present study is to evaluate the inhibitory potential of licorice extract and glycyrrhizin on cytochrome P450(CYP) in human liver microsomes. Methods : Using human liver microsomes, water extract of licorice and glycyrrhizin as an inhibitor were co-incubated with each probe drug representing selective CYP isoform activity. We measured relative metabolic activity in incubation condition compared to that with no extract of licorice using HPLC system. Results : Both water extracts of licorice and glycyrrhizin showed inhibitory effect on CYP-catalyzed reactions. CYP2C19 $(IC_{50}=126.7{\mu}g/ml)$ is most potently inhibited by water extract than other tested CYP isoforms$(IC_{50}>450{\mu}g/ml)$, but glycyrrhizin exhibited potent inhibition on CYP1A2$(IC_{50}=106.9{\mu}g/ml)$ followed by CYP2C9 and CYP2D6. Conclusion: These results indicate that water extract of licorice and glycyrrhizin have inhibitory potential on CYP-catalyzed reaction in human liver microsomes. But the mechanism of inhibition was slightly different between them Water extract of licorice mainly inhibited CYP2C19, and glycyrrhizin primarily inhibited CYP1A2. The inhibition by water extract of licorice and glycyrrhizin on CYP isoforms may cause drug interaction with co-administered drug leading to toxicity or treatment failure.

In Vitro Metabolic Stability of Moisture-Sensitive Rabeprazole in Human Liver Microsomes and Its Modulation by Pharmaceutical Excipients

Shan Ren,Mi-Jin Park,Aera Kim,이범진 대한약학회 2008 Archives of Pharmacal Research Vol.31 No.3

A reliable method to assess in vitro metabolic stability of rabeprazole and its modulation by Generally Recognized As Safe (GRAS)-listed pharmaceutical excipients was established in human liver microsomes. The metabolic stability of rabeprazole decreased as a function of incubation time, resulting in the formation of thioether rabeprazole via nonenzymatic degradation and enzymatic metabolism. Buffer type was also a determining factor for the degree of both nonenzymatic degradation and enzymatic metabolism. The net extent of enzymatic drug metabolism, obtained by calculating the difference in drug degradation between a microsomepresent reaction system and a microsome-free solution, was about 9.20 ± 0.67% in phosphate buffer and 2.27±1.76% in Tris buffer, respectively. Rabeprazole exhibited first-order kinetics in microsome-free solution but showed non-linear kinetics in the microsome-present reaction system. The maximal velocity, Vmax, in phosphate buffer was 5.07 µg mL-1 h-1 and the Michaelis- Menten constant, Km, was 10.39 µg mL-1 by computer-fitting to the classical Michaelis-Menten equation for pattern of time-dependent change in the substrate concentration. The intact drug and its thioether form were well resolved and successfully identified by HPLC chromatography and liquid chromatography mass spectroscopy (LC/MS). The metabolic stability of rabeprazole was also modulated by the presence of pharmaceutical excipients. Among the five pharmaceutical excipients tested, poloxamer 188 and Gelucire 44/14 had potentially inhibitory effects on rabeprazole metabolism in human liver microsomes (p < 0.05). A greater understanding of metabolic stability and its modulation by pharmaceutical excipients would be useful for optimizing the bioavailability of rabeprazole at the early formulation stages.

In Vitro Metabolic Stability of Moisture-Sensitive Rabeprazole in Human Liver Microsomes and Its Modulation by Pharmaceutical Excipients

Ren, Shan,Park, Mi-Jin,Kim, Ae-Ra,Lee, Beom-Jin 대한약학회 2008 Archives of Pharmacal Research Vol.31 No.3

A reliable method to assess in vitro metabolic stability of rabeprazole and its modulation by Generally Recognized As Safe (GRAS)-listed pharmaceutical excipients was established in human liver microsomes. The metabolic stability of rabeprazole decreased as a function of incubation time, resulting in the formation of thioether rabeprazole via nonenzymatic degradation and enzymatic metabolism, Buffer type was also a determining factor for the degree of both nonenzymatic degradation and enzymatic and enzymatic metabolism. The net extent of enzymatic drug metabolism, obtained by calculating the difference in drug degradation between a microsome-present reaction System and a microsome-free solution, was about $9.20\;{\pm}\; 0.67$% in phosphate buffer and $2.27{\pm}1.76$% in Tris buffer, respectively. Rabeprazole exhibited first-order kinetics in Microsome-free solution but showed non-linear kinetics in the microsome-present reaction system. The maximal velocity, $V_{max}$ in phosphate buffer was 5.07 ${\mu}g\;mL^{-1}\;h^{-1}$ and the michaelis-Menten constant, $K_m$, was 10.39 ${\mu}g\;mL^{-1}$ by computer-fitting to the classical Michaelis-Menten equation for pattern of time-dependent change in the substrate concentration. The intact drug and its thioether form were well resolved and successfully identified by HPLC chromatography and liquid chromatography mass spectroscopy (LC/MS). The metabolic stability of rabeprazole was also modulated by the presence of pharmaceutical excipients. Among the five pharmaceutical excipients tested, poloxamer 188 and Gelucire 44/14 had potentially inhibitory effects on rabeprazole metabolism in human liver microsomes (p < 0.05). A greater understanding of metabolic stability and its modulation by pharmaceutical excipients would be useful for optimizing the bioavailability of rabeprazole at the early formulation stages.

Role of cytochrome P450 and UDP-glucuronosyltransferases in metabolic pathway of homoegonol in human liver microsomes

Kwon, S.S.,Kim, J.H.,Jeong, H.U.,Ahn, K.S.,Oh, S.R.,Lee, H.S. 日本藥物動態學會 2015 DRUG METABOLISM AND PHARMACOKINETICS Vol.30 No.4

Homoegonol is being evaluated for the development of a new antiasthmatic drug. Based on a pharmacokinetic study of homoegonol in rats, homoegonol is almost completely eliminated via metabolism, but no study on its metabolism has been reported in animals and humans. Incubation of homoegonol in human liver microsomes in the presence of the reduced form of nicotinamide adenine dinucleotide phosphate and UDP-glucuronic acid resulted in the formation of five metabolites: 4-O-demethylhomoegonol (M1), hydroxyhomoegonol (M2 and M3), 4-O-demethylhomoegonol glucuronide (M4), and homoegonol glucuronide (M5). We characterized the cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes responsible for homoegonol metabolism using human liver microsomes, and cDNA-expressed CYP and UGT enzymes. CYP1A2 played a more prominent role than CYP3A4 and CYP2D6 in the 4-O-demethylation of homoegonol to M1. CYP3A4 was responsible for the hydroxylation of homoegonol to M2. The hydroxylation of homoegonol to M3 was insufficient to characterize CYP enzymes. Glucuronidation of homoegonol to M5 was mediated by UGT1A1, UGT1A3, UGT1A4, and UGT2B7 enzymes, whereas M4 was formed from 4-O-demethylhomoegonol by UGT1A1, UGT1A8, UGT1A10, and UGT2B15 enzymes.

Metabolism-mediated drug interaction potential of HS-23, a new herbal drug for the treatment of sepsis in human hepatocytes and liver microsomes

정현욱,이지영,권순상,김주현,김영묵,홍성욱,연성흠,이선미,조용연,이혜숙 대한약학회 2015 Archives of Pharmacal Research Vol.38 No.2

HS-23, an extract of the dried flower buds ofLonicera japonica, is a new botanical drug currently beingevaluated in a phase I clinical study in Korea for thetreatment of sepsis. The in vitro induction and inhibitionpotentials of HS-23 on the drug-metabolizing enzymesusing human hepatocytes and liver microsomes wereassessed to evaluate herb–drug interaction according tobotanical drug guideline and drug interaction guidance ofFDA. HS-23 slightly inhibited CYP2A6, CYP2B6,CYP2C9, CYP2C19, and CYP3A4 enzyme activities inhuman liver microsomes with IC50 values of 80.6, 160.7,169.5, 85.4, and 76.6 lg/mL, respectively. HS-23 showednegligible inhibition of CYP1A2, CYP2C8, CYP2D6,UGT1A1, UGT1A4, UGT1A9, and UGT2B7 activities inhuman liver microsomes. Based on these results, HS-23may not inhibit the metabolism of CYP2A6, CYP2B6,CYP2C9, CYP2C19, and CYP3A4-catalyzed drugs inhumans. HS-23 did not affect the mRNA expression ofCYP1A2, CYP2B6, and CYP3A4 after 48 h treatment atthree concentrations (0.5, 5, and 50 lg/mL) in three independenthuman hepatocytes, indicating that HS-23 has noeffect on herb–drug interactions that up- or down-regulateCYP1A2, CYP2B6, and CYP3A4. These results indicatethat the administration of HS-23 in human may not causeclinically relevant inhibition and induction of these cytochromeP450 (CYP) and UDP-glucuronosyltransferase(UGT) enzymes and HS-23 may be promising therapeuticagent for treatment of sepsis.