초록 ▼

신물질 개발의 초기단계인 lead selection과 optimization에서 최적의 대사 및 체내동태 특성을 평가할 수 있는 기술을 개발하고자 하였으며 다음과 같은 결과를 얻었음
- 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 확인과 MS/MS spectrum으로 구조 유추가 가능한 방법을 확립
- Meta

신물질 개발의 초기단계인 lead selection과 optimization에서 최적의 대사 및 체내동태 특성을 평가할 수 있는 기술을 개발하고자 하였으며 다음과 같은 결과를 얻었음
- 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 확인과 MS/MS spectrum으로 구조 유추가 가능한 방법을 확립
- Metabolic profile과 구조분석: Product ion mass spec., NMR, stable isotope labeling등 대사체의 구조를 규명하기 위한 다양한 approach 방법을 확립하였으며 pyronardidne등 10여종 약물의 대사 profile 규명
- Radio-labeled compound를 이용하여 LADME 연구를 수행 기반을 확립하였으며, 전신 분포 규명을 위한 rat whole body autoradiography 방법을 확립하고 L$\underline{\space\space}$FMAU, telemsteine의 autoradiogram을 획득 조직분포를 규명.
- 미량 약물의 분석을 위하여 GC/MS, HPLC/MS/MS, 그리고 switching HPLC 방법을 도입하여 정량이 어려운 steroid, 당화합물, 그리고 tizanidinel등의 극미량 분석 방법을 개발 확립하여 실제 PK 연구에 적용
- In vitro Caco-2 permeability 평가 방법의 확립과 물리화학적 특성에 따른 Papp 값의 변화, 그리고 흡수율의 상관성을 규명하였으며, 뇌질환치료제의 BBB 투과성 평가를 위한 채 -culture system을 개발하여 평가하고자 함
- Human P450 8종의 isozyme을 E. coli에 reductase와 동시 발현하여 이를 이용한 flavonoid들의 활성 억제의 selectivity를 규명하고 체내에서 약물 상호작용 평가를 위하여 cassette analysis를 이용한 CYP 효소 억제 정도를 동시 평가하는 방법을 개발
- Early stage에서 in vitro 결과를 이용하여 human PK를 예측하는 방법을 개발 중이며, 또한 urinary 대사체의 분석과 결과의 통계분석을 이용한 독성예측 평가방법을 개발하여 적용성을 평가중에 있음.

Abstract ▼

Desirable metabolic and pharmacokinetic properties of drug candidates are required to be approved as new drugs. During last 10 years, major cause of dropout of newly developed drugs in clinical stages are pharmacokinetic and metabolic issues. Therefore, the selection and optimization of candidates i

Desirable metabolic and pharmacokinetic properties of drug candidates are required to be approved as new drugs. During last 10 years, major cause of dropout of newly developed drugs in clinical stages are pharmacokinetic and metabolic issues. Therefore, the selection and optimization of candidates in early stage of drug discovery stage is prerequisite for successful drug development. The purpose of present study was to establish rapid metabolic/absorption screening system for the selection and optimization of hit and/or lead compounds. Secondly, development of systemic approach to elucidate the structure of metabolites was aimed to identify the metabolic hot-spot. Thid purpose was to construct the base for applying radiolabeling to preclinical ADME 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 and humn 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 incubtion with rat liver microsomes were pooled into the designed cassette groups and analyzed by HPLC/electrosparay (ESI) ITMS in full-scan mode. The metabolic stability of the drugs was determined by comparing their signals after incubation for 0 and 30 min, respectively. The metabolic stability of examined dopamine receptor antagonists was in the range of 9.9-84.4$\%$. In additon, present cassette analysis allowed simultaneous detection of metabolites formed during the same incubation without having to reanalyze the samples. 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. Metabolic profiles and characterization of structure of metabolites are important to understand the fate of the compounds and to optimize the structures. Several approaches were developed: 1) product ion mass spectrometry coupled with NMR spectroscopy, 2) tracing with radiolabeled compounds, and 3) application of stable isotope labeling to differentiate the isobaric metabolites. By using these methods, metabolic profiles of mor than 10 compounds were characterized including pyronaridine and DDB derivatives. Determination of trace amounts of drugs or metabolites in biological species is also very important. For the development of more sensitive and selective analytical method for the analysis of drugs, column-switching HPLC couple with UV detection, LC/MS/MS method, and GC/MS method have been developed abd their application has been decribed. By using these technique detection limit could be lowered to subnano gram/ml level. 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 $\textohm$/cm$^2$ and mannitol permeability was reduced down to 0.25$\textohm$/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. 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 vivo plasma concentration of drugs using PBPK model has been developed. Also, the analysis of total urinary metabolitea and principal component analysis of the data was extremely useful to predict the hepatic toxicities of the chemicals. The application of the above-described methods to new drug development will facilitate the development process and increase the success rate.

목차 Contents

• 제1장 신속 in vitro 대사 안전성 평가 및 대사체 구조 규명...20
• 제2장 개발 약물의 대사 profiling 및 구조분석 연구...48
• 제3장 방사성 동위원소를 이용한 ADME 및 autoradiography...122
• 제4장 새로운 분석 방법의 개발 및 응용...144
• 제5장 약물 상호작용 평가를 위한 human P450 효소발현 및 in vitro inhibition 평가방법 개발...198
• 제6장 소장과 BBB 투과성 평가의 개발 및 응용...230
• 제7장 In vitro 결과를 이용한 PK 예측 및 대사체분석을 이용한 독성 예측...274