초록 ▼

- HPLC/MS/MS를 이용한 rapid In vitro metabolic stability 평가기술 개발: 다 약물 동시 분석과 data analysis 방법개발로 기존 평가 방법 보다 분석 시간을 10배 단축하였으며, single analysis에서 대사체 확인까지 기능하였음.
- MS chromatogram에서 대사체 유추와 MS/MS tandem mass spectrum으로부터 대사 구조 유추 방법을 확립하여 20여 종 화합물에서 생성되는 대사체 구조 해석
- In vitro Caco-2 세포 배양 조건과 per

- HPLC/MS/MS를 이용한 rapid In vitro metabolic stability 평가기술 개발: 다 약물 동시 분석과 data analysis 방법개발로 기존 평가 방법 보다 분석 시간을 10배 단축하였으며, single analysis에서 대사체 확인까지 기능하였음.
- MS chromatogram에서 대사체 유추와 MS/MS tandem mass spectrum으로부터 대사 구조 유추 방법을 확립하여 20여 종 화합물에서 생성되는 대사체 구조 해석
- In vitro Caco-2 세포 배양 조건과 permeability 평가 기반 확립 (HPLC/MS 방법을 이용한 rapid analysis) 기존의 20여개 화합물의 Papp 값을 구하고 physio-chemical property와의 관련성을 평가하였으며 PSA 값이 Caco-2 세포 permeability 정도와 상관성이 높음을 입증
- Human P450 6종의 isozyme을 E. coli에 reductase와 동시 발현하여 이를 이용한 flavonoid들의 활성 억제의 selectivity를 규명하고 또한 대사에 관여하는 P450 동정 연구에 활용 기반을 구축
- 미량 약물의 분석을 위하여 GC/MS, HPLC/MS/MS, 그리고 switching HPLC 방법을 도입하여 정량이 어려운 steroid, 당화합물, 그리고 taxol등의 극미량 분석 방법을 개발 확립.
- Radio-labeled compound를 이용하여 L-FMAU의 ADME 연구를 수행 기반을 확립하였으며, 전신 분포 규명을 위한 rat whole body autoradiography 방법을 확립하고 L_FMAU, telemsteine의 autoradiogram을 획득 조직분포를 규명하였음
- HPLC/MS/MS, GC/MS등의 방법을 이용하여 eperisone과 DDB등의 in vivo metabolic Profile을 규명하고 또한 대사체의 구조를 확인
- 신규화합물의 대사 및 체내동태 특성을 in vitro에서 평가하는 체계를 구축하고, 전임상 ADME 연구 기반을 확립하였음

Abstract ▼

Drug metabolism and pharmacokinetic properties of drug candidates are determinant factors in successful new drug development. During last 10 years, major cause of dropout of newly developed drugs in clinical stages are pharmacokinetic and metabolic issues. Therefore, selection and optimization of ca

Drug metabolism and pharmacokinetic properties of drug candidates are determinant factors in successful new drug development. During last 10 years, major cause of dropout of newly developed drugs in clinical stages are pharmacokinetic and metabolic issues. Therefore, selection and optimization of candidates in early drug discovery stage is prerequisite for successful drug development. The purpose of present study is to establish rapid metabolic/absorption screening method and to construct the base for applying radiolabeling to preclinical ADIVIE study. In vitro metabolic stability of dopamine D3/D4 receptor antagonists and identification of their metabolites by high performance liquid chromatography (HPLC) coupled with ion trap mass spectrometry (ITMS) were done in rat liver microsomes. The compounds were divided into three cassette groups for rapid quantitative analysis of multiple drugs and simultaneous detection of their metabolites. The samples from incubation with rat liver microsomes were pooled into the designed cassette groups and analyzed by HPLC/electrospray (ESI) ITMS in full-scan mode. The metabolic stability of the drugs was determined by comparing their signals after incubation for 0 and 30 mm, respectively. The metabolic stability of examined dopamine receptor antagonists was in the range of 9.9-84.4%. In addition, present cassette analysis allowed simultaneous detection of metabolites formed during the same incubation without having to reanalyze the samples. The metabolites were first characterized by nominal mass measurement of the corresponding protonated molecules. Subsequent multi-stage tandem spectrometry ($MS^n$) on the ion trap instrument allowed characterization of structure of the detected metabolites. N,O-dealkylation and ring hydroxylation reaction were identified to be major metabolic reaction in piperazinylalkylisoxazole derivatives. These results suggested that present approach is useful for rapid evaluation of metabolic stability and structural characterization of metabolites within a short period in new drug discovery. The culture condition of Caco-2 cells was established and apply this system to evaluate in vitro permeability study. After 17 day culture, TEER value of monolayered cells could reach to over 600${\Omega}$/$cm^2$ and mannitol permeability was reduced down to 0.25${\Omega}$/$cm^2$. In vitro permeability of D3/D4 receptopr antagonists was evaluated using this system. The permeability of these compounds varied depending on the structure and permeability coefficient was between 0.9 and 24.0. For the rapid evaluation of permeability, cassette analysis of mixtures was done using HPLC/MS/MS spectrometry. The permeability coefficients of compounds obtained by cassette analysis was well correlated with those obtained by single individual analysis. The permeability depends on physio-chemical properties of compounds. Polar surface area (PSA) and Log P value were calculated and simulated with experimental results. The permeability of chemicals was mostly goverened by PSA value and correlation coefficient between PSA and Caco-2 experimental value was over 0.7. The hypernated analytical technique is essential for pharamcokinetics study and tracing drug metabolites. HPLC/MS/MS product ion monitoring, column-switching HPLC, and gas chromatography/mass spectrometic selective ion monitoring methods were developed for sensitive analysis of drugs such as medroxyprogesterone acetate, taxol, and tizanidine. By using these technique detection limit could be lowered to subnano gram/ml level. We also successfully apply radio-labeling to ADME experiment and whole body autoradiographic technique was developed. In conclusion, the methods and techniques required for lead selection and optimization in drug discovery process and preclinical ADME evaluation.

목차 Contents

• 표지...1
• 제출문...2
• 보고서 초록...3
• 요약문...4
• SUMMARY...11
• CONTENTS...13
• 목차...14
• 제1장 신속 in vitro 대사 안전성 평가 및 대사체 구조 규명...15
• 제2장 Caco-2 세포를 이용한 소장 흡수 연구...56
• 제3장 P450 발현 및 inhibition 연구...79
• 제4장 새로운 분석 방법의 개발 및 응용...97
• 제5장 Radio-labeled compound를 이용한 ADME 연구...127
• 제6장 대사체 구조 및 대사 경로 규명...147