In vitro Metabolism of Herbicide Flucetosulfuron by Human Liver Microsomes and Artificial Gastrointestinal Juices

Abstract

본 연구에서는 국내에서 최초로 개발된 신규 sulfonylurea 제초제인 flucetosulfuron 의 인체간마이크로좀(Human liver microsomes

HLMs)과 인공소화액(Artificial gastrointestinal juices)에 의한 in vitro 생체대사연구를 수행하였다. Flucetosulfuron 은 threo 와 erythro 인 부분입체이성질체로 구성되어 있기 때문에, 각각의 분리과정을 거쳐서 99.7% 순도의 threo-flucetosulfuron 과 99.8% 순도의 erythroflucetosulfuron 을 확보하여 대사연구에 적용하였다. HLMs 을 이용한 in vitro 대사시험에서 생성된 대사물은 유일하게 M1 이 생성되었고, M1 은 threo-M1(TM1

(1S,2S)-1-(3-(N-(4,6-dimethoxypyrimidin-2-ylcarbamoyl)sulfamoyl)pyridin-2-yl)-2-fluoropropyl-2-methoxyacetat)과 erythro-M1 (EM1

(1R,2S)-1-(3-(N-(4,6- dimethoxypyrimidin-2-ylcarbamoyl)sulfamoyl) pyridine-2-yl)-2-fluoropropyl-2-methoxyacetate)이었다. TM1 과 EM1 은 각각 threo-flucetosulfuron 과 erythro-flucetosulfuron 으로부터 생성되었으며, LC-MS/MS 와 NMR 분석 및 합성한 TM1 과 EM1 의 Cochromatography 방법을 통해 확인하였다. TM1 과 EM1 은 가수분해를 통해서 생성되며, 이를 담당하는 효소는 cytochrome P450 이나 flavin-containing monooxygenase 가 아닌 esterases 임을 확인하였다. 최적화된 조건에서 HLMs 의 동력학시험을 실시하여 flucetosulfuron 과 대사물들을 정량분석한 결과, threoflucetosulfuron 의 Vmax (nmol/min/mg HLMs)와 Km (μM)과 CLint (Vmax/Km (μL/min/mg HLMs)) 는 각각 134.38 과 1798.53, 51.2 였고, erythro-flucetosulfuron 은 각각 151.41 과 3957.37, 48.02 로 나타났으나, HLMs 의 대사연구에서 두 이성질체의 구조적 차이에 따른 유의성있는 차이를 볼 수 없었다. Esterases 의 선택적 저해시험을 통해 flucetosulfuron 의 가수분해에 직접적으로 관여하는 효소는 Carboxylesterases 와 Cholinesterases 임을 확인하고, 각각의 human acetylcholinesterase (AChE)와 human butyrylcholinesterase (BChE), 그리고 3 종의 재조합 human carboxylesterases (CES: CES1b 와 CES1c, CES2)의 효소들을 이용한 대사시험을 실시하여, HLMs 대사시험과 동일한 M1 (TM1 과 EM1)이 생성됨을 검증하였다. 따라서, 상기의 효소들에 의한 동력학시험을 실시하고, flucetosulfuron 과 대사물들의 정량분석을 하였다. CES2 의 CLint (Vmax /Km (μL/min/mg HLMs)는 threo-와 erythro-flucetosulfuron 에서 각각 16.98 과 17.43 수준으로, 시험한 재조합 CES 효소들 중에 가장 flucetosulfuron 에 대한 높은 가수분해활성을 보여주었다. Threoflucetosulfuron 에 대한 AChE 와 BChE 의 Vmax 는 erythroflucetosulfuron 보다 각각 2.0 배와 3.8 배로써, BChE 에서 더 큰 차이를 보였다. 또한, BChE 에서의 threo-flucetosulfuron 의 CLint 는 4.3 으로, 0.87 을 보인 erythro-flucetosulfuron 보다 5 배수준으로 빠르게 대사됨을 보여주었다. CES1b 와 CES1c, CES2, AChE 와는 달리, BChE 에서는 flucetosulfuron 이성질체들의 선택적인 대사차이가 유의성있게 나타남을 알 수 있었다. 인공소화액인 타액, 위액과 장액을 이용한 in vitro 대사시험에서는 HLMs 보다 다양한 대사물이 생성되었다. 인공타액에서는 분해가 없었으나, 인공위액에서는 설포닐유레아결합과 에스터결합의 분해산물들인 2-(2-fluoro-1-hydroxypropyl)pyridine-3-sulfonamide (M2)와 4,6-dimethoxypyrimidin-2-amine (M3), 2-fluoro-1-(3-sulfamoylpyridin-2-yl)propyl 2-methoxyacetate (M4)가 관찰되었고, 인공장액에서는 M1 이 주요대사물로 관찰되었다. Flucetosulfuron 과 대사물들의 정량분석결과, 인공위액에서 85%이상의 빠른 분해를 보였고, 장액에서는 18%의 분해를 보였으나, flucetosulfuron 의 이성질체간의 분해의 양상의 차이는 없었다. Flucetosulfuron 이 경구를 통한 체내 유입될 경우, 인공소화액에서 분해가 빠르게 진행되므로, 실제 체내 흡수량이 미미할 것으로 판단되었다. 본 연구결과, HLMs 과 인공소화액에 의한 플루세토설퓨론의 대사양상이 제안되었다.

To investigate in vitro metabolism of herbicide, flucetosulfuron, with human liver microsomes (HLMs) and artificial gastrointestinal juices (GIs), threo- and erythro-isomers of flucetosulfuron were prepared as 99.7% and 99.8% purity, respectively. In vitro metabolism of threo- and erythro- flucetosulfuron by HLMs produced M1, threo-metabolite (TM1

(1S,2S)-1-(3-(N-(4,6-dimethoxypyrimidin-2-ylcarbamoyl)sulfamoyl)pyridin-2-yl)-2-fluoropropyl-2-methoxyacetate) from threo-flucetosulfuron and erythro-metabolite (EM1

(1R,2S)-1-(3-(N-(4,6-dimethoxypyrimidin-2-lcarbamoyl)sulfamoyl)pyridin-2-yl)-2-fluoropropyl-2-methoxyacetate) from erythro-flucetosulfuron, respectively. TM1 and EM1 were identified by LC-MS/MS and NMR and unambiguously confirmed by cochromatographic method by comparison with the synthesized authentic TM1 and EM1, having 99.7% and 96.5% purity, respectively. Hydrolytic metabolism of flucetosulfuron was proved to be mediated not by P450s or FMOs but by esterases. After optimization of reaction time, HLMs concentration, and flucetosulfuron concentration, kinetics parameters of each flucetosulfuron isomers were obtained from Michaelis-Menten plot. The estimated Vmax (nmol/min/mg HLMs) and Km (μM) values were calculated 134.38 and 2798.53 for threo-flucetosulfuron and 151.41 and 3957.37 for erythro-flucetosulfuron, respectively. The intrinsic clearance (CLint = Vmax /Km (μL/min/mg HLMs)) values of the formation of TM1 from threo-flucetosulfuron and EM1 from erythro-flucetosulfuron were 51.20 and 48.02, respectively. The CLint values from flucetosulfuron isomers by HLMs did not show any significant difference. Inhibition test with selective esterases inhibitors indicated that the metabolizing esterases in hydrolysis of flucetosulfuron are carboxylesterases and cholinesterases. Esterases kinetics with human recombinant carboxylesterases (CES1b, CES1c and CES2), human acetylcholinesterase (AChE), and human butyrylcholinesterase (BChE) demonstrated that the metabolism of flucetosulfuron isomers is mediated by these esterases with the same results from the specific esterase inhibition. The CLint values of CES2 for the formation of TM1 and EM1 showing the highest activity among the tested esterases were 16.98 and 17.43. The CLint values of CES1b and CES1C were 3.99 and 4.5 for threo-flucetosulfuron and 2.59 and 3.07 for erythro-flucetosulfuron. CLint value of AChE for threo-flucetosulfuron was 2.02, also showing not much of a difference to 2.32 for erythro-flucetosulfuron. However, CLint value of BChE for threo-flucetosulfuron was 4.43, showing 5-fold higher than 0.87 for erythro-flucetosulfuron, with the 3.8-fold higher Vmax values of BChE for threo-flucetosulfuron than erythro-flucetosulfuron. The differences of the kinetic parameters between flucetosulfuron isomers indicated that there is a possibility of stereoselcetive metabolism between threo- and erythro-flucetosulfuron by esterase such as BChE. The investigation on in vitro metabolism of flucetosulfuron by artificial GIs (saliva, gastric juice, intestinal juice) showed that there are not significant differences in the degradation patterns between flucetosulfuron isomers in GIs. Flucetosulfuron was observed to be stable in saliva, while about 85% of flucetosulfuron in the reaction with gastric juice was rapidly degraded and produced metabolites, 2-(2-fluoro-1-hydroxypropyl)pyridine-3-sulfonamide (M2), 4,6-dimethoxypyrimidin-2-amine (M3), and 2-fluoro-1-(3-sulfamoylpyridin-2-yl)propyl 2-methoxyacetate (M4), indicating the occurrence of the hydrolysis of sulfonylurea-bridge and ester bond of flucetosulfuron. In the reaction with intestinal juice, about 18% of flucetosulfuron was degraded and M1 was observed as only metabolite of flucetosulfuron, suggesting the breakdown of ester-bond of flucetosulfuron. During these studies, there was not any chiral conversion between flucetosulfuron isomers or metabolite isomers. On the basis of these results, the metabolic pathway of flucetosulfuron in HLMs and artificial gastric juices is proposed.