보고서 정보
주관연구기관 |
한국과학기술연구원 Korea Institute Of Science and Technology |
보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 1998-05 |
주관부처 |
보건복지부 [Ministry of Health & Welfare(MW)(MW) |
연구관리전문기관 |
한국보건산업진흥원 Korea Health Industry Development Institute |
등록번호 |
TRKO201400018371 |
DB 구축일자 |
2014-11-29
|
초록
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IV. 연구개발결과
Hurnan rnetabo1isrn과 대사관여 P450효소 연구를 위하여 10 여종의 human liver 를 확보하여 각각의 대사특성을 규명하였다. 개체간의 P450 isozyme 분포와 효소활성도는 개체간와 차이를 보여 주었으며, Mianserin 대사profile은 HPLC와 GCjMS를 이용하여 정량 및 구조 분석을 하였으며 authentic cornpound와 비교하여 확인한 결과 8-hydroxymianserin, desmehty mianserin, 그리고 mianserin N-oxide로 대사
IV. 연구개발결과
Hurnan rnetabo1isrn과 대사관여 P450효소 연구를 위하여 10 여종의 human liver 를 확보하여 각각의 대사특성을 규명하였다. 개체간의 P450 isozyme 분포와 효소활성도는 개체간와 차이를 보여 주었으며, Mianserin 대사profile은 HPLC와 GCjMS를 이용하여 정량 및 구조 분석을 하였으며 authentic cornpound와 비교하여 확인한 결과 8-hydroxymianserin, desmehty mianserin, 그리고 mianserin N-oxide로 대사 되었으며 인체 특이 대사체는 검출되지 않았다. Dog의 경우 human과 대사profile 이 가장 유사하였다 In vitro method에 따른 차이는 microsomal fraction, liver slice, 그리고 hepatocytes culture를 이용하여 mianserin 01 8-hydroxymianserin, desmehty mianserin, 그리고 mianserin N-oxide로 대사profile 비교시 liver slice가 in VIVO와 가장 유사한 profile을 나타내었다. 약물대사 관여 효소 확인, enzyme kinetics를 이용한 drug-drug interaction, 그리고 catalytic 특성 연구를 위하여 CYPIA2, CYP2CI0, CYP3A4, CYP2D6, 그리고 CYP2El gene을 PCW plasmid 에 insertion 시킨후 E. coli DH5a 에서 발현시킨 후 column chromatography를 이용하여 정제하였다. Immuno inhibition 실험을 위하여 토끼에서 각각의 lsozyme에 대한 antibody를 생산 하였다. 이들을 이용하여 실험결과 mianserin 대사는 multienzyme reaction으로 CYPIA2와 CYP3A4 효소가 desmethylmianserin과 mianserin-N-oxide, 그리고 8-hydroxy mianserin 대사에 판여하고 CYP2D6가 8-hydroxymianserin 대사에 관여 함을 확인하였다. Intrinsic clearance는 대사체 모두 0.001-0.01 ml/min/mg protein으로 in vivo metabolic clearance가 비교적 낮올 것으로 추정되며 인체 P450에 의한 intrinsic clearance 가 dog보다 높은 것으로 나타났다 Human P450의 catalytic 특성으로 대사에 가장 많아 관여하는 CYP3A4효소의 aflatoxin Bl 대사 kinetics로 two binding site 임을 증명하였으며 CYPIA2효소 억제와 flavonoids의 구조 상관관계를 확립하였다. 감 보호제로 사용되는 DDB의 경우 혈중농도가 아주 낮다. DDB는 in vitro microsome과 반응시 션속히 demethytation 과 methylene dioxy ring cleavage가 일어난 대사체(M1-M7) 대사되며 rat과 상이한 대사 profile울 보여주었다. 각대사체를 mass, NMR spectroscopy로 구조 분석을 하였으며 향후 kinetic study를 수행하여 metabolic inhibition 수란으로 보다 우수한 약효를 유발하는 방법이 개발될 것으로 사료된다.
약물을 rat, mouse등외 동물에 투여할 경우에 ①간조직중의 microsomal epoxide hydr’olase (mEH) 의 유도증겨 현상,②조척중의 glutathione S-transferases (GST) 역 유도증가를 연구하기 위하여 효소발현 및 유전자 발현수준에서의 정량방법을 확립하였다. 이러한 해톰화 효소를 단백질 및 유전자 발현수준에서 관찰하는 방법을 확립하거 위한 일차적인 목표로써 mEH 및 GST 의 주요 subunit 인 Ya, Ybl, Yb2, Yc1 및 Yc2유전자의 coding region을 polymerase chain reaction으로 종폭하여 cloning 하였다. Alkyl sulfide 계 화합물 및 Thiazole계 화합물이 Microsomal Epoxide Hydrolase 몇 Glutathione Glutathione S-Transferase Ya, Yb1, Yb2, Yc1, Yc2 유전자 발현에 미치는 효과는 랫드 모델에서 관찰하였다. 기초화합물을 중심으로 이들 효소(계)에 미치는 유전자발현효과를 연구하여 약물개발에 필요한 자료로 활용할 수 있을 것으로 생각되다, mEH 및 GST유도에 관여하는 moiety 에 관한 체계적인 구조활성연구가 이루어진 바 없다. 본 연구를 통하여 많은 약물의 모핵 구조로 들어 있는 heterocyle 화합물 중 5,6 각환 화합물과 Benzene 이 융합된 구조의 heterocycle 화합물이 이들 해독화효소의 발현에 미치는 효과를 관찰하였다. 해독화대사 효소의 발현에 관련된 기초연구자료 확보 및 관할된 연구기술의 정립을 위하여 랫드모델에서 mEH 와 GST 의 각 subunit 의 유전자발현과 관련하여 benzene-fused heterocycles 의 효과를 model화합물로써 검정하고/ 약물의 효과를 mouse 에서 비교검토하였다. Methyl기가 도입된Thiazole을 모델화합물로 이용하여 이들 화합물에 의한 rat 간조직 중의 microsomalepoxide hydrolase (mEH), glutathione S-transferase (GST) 발현을 정량하고/ 관련된 유전자 발현기전을 연구하여 methy1thiazoles 및 thiazole 유도채의 분자구조특정/P450 에 의한 대사활성화기전, bioactivation에 따른 간독성발현을 이들 해독화효소유도와 연계 하여 규명하였다. 나타남을 알수 있었다. 또한 란소프라졸과 사이크로스포린 및 데오필린과 병용투여시 약물상호작용을 연구한 결과는 사람에서의 결과 (문헌에서 란소프라졸의 체내동태연구 결과 사람에서 개체차가 매우 크게 나타났는데, 실험동물에서도 개체차가 크게유추)와 잘 일치하였고, 또한 실험동물 (랫트)콜 이용하여 한약과 데오필린 투여시 데오필린의 약동학에 미치는 한약의 영향도 환자에서 얻은 케이스보고와 잘 일치 함을 알 수 있었다.
Abstract
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In vitro drug metabolism, the mechanism of enzyme induction, and pharmacokinetic studies were done in order to establish the methods to apply the drug metabolism and pharmacokintic tools to potential drug development.
Human liver samples showed a marked interindividual differences in isozyme act
In vitro drug metabolism, the mechanism of enzyme induction, and pharmacokinetic studies were done in order to establish the methods to apply the drug metabolism and pharmacokintic tools to potential drug development.
Human liver samples showed a marked interindividual differences in isozyme activities. Mianserin, an antidepressant agent, was metabolized to 8-hydroxymianserin, desmethylmianserín, and mianserin N-oxide by microsomes and their structure were identified on the basis of mass spectroscopy and HPLC cochromatography. Human specfic metabolites were not identified and metabolic profile in dogs was most simìlar to that in human. The metabolism of mianserin varied on in vitro system and liver slice showed the representative pattern similar to physiological settings. Human CYPIA2, CYP2CI0, CYP3A4, CYP2D6, and CYP2El genes were cloned in pCW vector and overexpressed in E. coli in order to obtain purified enzymes required to characterize metabolism kinetics and to identify P450 enzyme inviolved in the metabolism of mianserin. Anti P450 antobodies were also produced by injecting proteins to rabbits. CYPIA2 and CYP3A4 were identified to be involved in the metabolism of mianserin and CYP2D6 was partially involved in the generation of 8-hydroxymianserin. Intrinsic clearance of metabolites was in range of 0.001-0‘01 mljminjmg .protein, suggesting that in vivo metabolic c1earance would occur relatively at low rate.
Two binding site in CYP3A4 was characterized by analyze the kinetics of aflatoxin Bl metabolism and structure-related inhibition of CYP1A2 activity by naturally occurring flavonoids was estabìlshed. The hepatoprotective agent DDB, showed low plasma concentration. DDB was extensively metabolized by human liver microsomes. Seven metabolites were identified in in vitro incubations and some of the structure of metabolites were characterized by mass and NMR spectral analysis. Demethylation in the methoxy and carboxymethyl group and ring cleavage of methylenedioxy ring was predominantly occurred. The kinetic analysis and identification of P450 enzyme involved in the metabolism of DDB would be required to develop clinically useful methods to íncrease the plasma concentratIon.
The expression of glutathione S-transferases (GSTs) in response to organosulfur compounds inc1uding allyl disulfide(ADS), allyl sulfide (AS), allyl ether (AE), propyl disulfide (PDS), propyl sulfide (PS), propyl ether (PE) and butyl sulfide (BS) as prototypic organosulfur compounds was studied. Northem blot analyses revealed that ADS and AS concomitantly stimulated GST Ya, Ybl and Yc2 gene expression, whereas PS increased the levels of Ya, Ybl and Yb2 mRNA but not Y c2 mRNA levels. The expression of GST subunit Yc2 in response to these compounds might be associated with hepatoprotective effects.
The effects of thiazole (TH), benzothiazole (BT) and benzothiadiazole (BZ) on the expression of hepatic glutathione S-transferases (GSTs) Ya, Yb1, Yb2, Yc1, Yc2, and microsomal epoxide hydrolase (mEH) genes were compared in animals. TH treatment resulted in 4- to 24-fold increases in GST Ya mRNA levels at 24 hr post-treatment‘GST Ya mRNA levels were elevated in a time-dependent manner. BT was a moderate inducer of GST Ya with a maximal 18-fold increase being observed, whereas BZ treatment caused a transient increase in GST Ya mRNA. TH was effective in modulating both constitutive and inducible GST gene expression. BT or BZ was much less effechve in increasing the expression of GST subunits. RNA and Westenl blot analyses revealed that the levels of major GST were differentially increased after treatment with these thiazoles, exhibiting a rank order of GST expression of TH>BT>BZ. mEH expression by these compounds appeared to be consistent with that of GST Ya. These results corroborate evidence that the thiazoles differentially stimulate GST or mEH genes with TH being the most efficacious, that thiazoles with carbocyclic ring are much less effective in increasing GST or mEH levels than TH, and that the changes ín these GST and mEH levels are primarily associated with increases in their mRN A levels.
The effects of methylthiazoles on the gene expression were studied with the aim of elucídating the molecular basis of gene expression and hepatotoxicity of the agents ín association with cytochrome P450一mediated metabolic activation.
Treatment of rats with methylthiazoles including 4-methylthiazole (4-MT), 5-methylthiazole (5-MT), 4,5-dimethylthiazole (DT), 2,4,5-trimethylthiazole (TT), 4-rnethyl-5-vinylthiazole (MVT) and thiazole (TH) resulted in differential increases in mEH mRNA level. 4-MT and TH were effective in stimulating the mEH gene. 5-MT, DT and TI failed to increase the mEH mRNA level after 3 day of treatment. GST Ya mRNA level was also sìgnificantly elevated after 4-MT or TH treatment, as compared to control, whereas the gene expression was affected much less by thiazoles with methyl groups at both 4 and 5 positions, Thus, addition of methyl group(s) to thíazole at 5 position resulted in decreases in efficacy for the mEH and GST induction. Correlation study and induction experiments provided evidence that metabolíc activation of certain thiazoles by P450 3Al/2 and possibly P450 lA2 may contribute to increases of hepatic mEH and GST mRNA levels. These results support the conc1usion that induction of mEH and GST by thiazoles is mediated with metabolic activation at 5 position, which precedes transcriptional activation of mEH and GST genes, and that reduction of P450 levels by 4-MT is associated with its hepatotoxicity.
Taken together, these results showed that N- or S-containing agents differentially alter the expression of the detoxifying genes, which affects xenobiotic-induced tissue toxicity in association with metabolic activation.
It is very useful for clinical applications of new drugs to humans to predict the pharmacokinetic characteristics of some investigational drugs in humans from the resu1ts of preclinical animal study. And in present study, we tried to compare the pharmacokinetic characteristics of lansoprazole and theophylline in humans and rats, and also check the pattems of drug interactions of these drugs with other medicines. In the case of drug interactions, we selected 4 topics, these were 1) lansoprazole and cyclosporin, 2) lansoprasole and theophyl1in, 3) theophylline and Korean traditional herbal medicines, and 4) theophylline and corticosteroid 一methylprednisolone. We used S.D. male rats, which were the ranges of 260 - 300 gram of body weight when these animals used to pharmacokinetic studies. From the 4 experiments, we found some kinds of useful informations. First, lansoprazole has a large individual differences in humans and animals. Second, cyclosporin has effects on the absorptions of lansoprazole, but theophylline on the metabolism of lansoprazole in rats. Third, Korean traditional herbal medicine, Hesochongi-Tang, has effects on the eliminations of theophylline in rats, that is, the dearance of theophylline in rats was increased at the case of coadministration with Hesochongi-Tang.. And at last, methylprednisolone has no effects on the pharmacokinetics of theophylline in rats. From the results of these studies, it was noted that the pharmacokinetic characteristics of drug interactions of investigational drugs in humans might be predictable from the animal studies. And it may be very useful to apply these animal scale-up study to check the drug interactions of Korean traditional herba1. medicins and frequently prescribed ethical drugs in Korea.
목차 Contents
- 표지 ... 1
- 제출문 ... 3
- 요약문 ... 5
- SUMMARY ... 11
- CONTENTS ... 15
- 목 차 ... 17
- 제 1 세부과제 In vitro 대사 기법 및 human tissue 에 의한 대사 ... 19
- 제 1 장 서 론 ... 21
- 제 2 장 국내외 연구개발 현황 ... 25
- 제 3 장 연 구개 발 수행 내 용 및 결 과 ... 27
- 제 4 장 연구개발목표 달성도 및 기여도 ... 103
- 채 5장 연구계발 결과의 활용계획 ... 105
- 제 6장 참고문헌 ... 107
- 제 2 세부과제 약물의 동태연구기술확립을 위한 種특이성연구: Microsomal epoxide hydrolase 와 Glulalhione S-transferases 의 발현연구 ... 111
- 제 1 장 서론 ... 113
- 제 2장 국내·외 기술개발 현황 ... 115
- 제 3장 연구개발수행 내용 및 결과 ... 117
- 제 4 장 연구개발목표 달성도 및 대외기여도 ... 161
- 제 5 장 연구개발결과의 활용계획 ... 163
- 제 6 장 참고문헌 ... 165
- 제 3 세부과제 체내동태 및 In Vitro - In Vivo 상관성에 관한 연구 ... 167
- 제 1 절 서론 ... 169
- 제 2 절 국내·외 연구현황 ... 173
- 제 3절 연구개발 수행내용 및 결과 ... 175
- 제 4 절 연구개발 목표 달성도 및 대외 기여도 ... 219
- 제 5절 연구개발 결과의 활용계획 ... 221
- 제 6 절 참고문헌 ... 223
- 최종보고서 요약 (초록) ... 227
- 끝페이지 ... 230
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