초록 ▼

본 연구에서는 고지혈증 치료제 개발을 위한 cholesterol ester transfer protein (CETP) 차단제 후보물질의 전임상연구를 수행하고자, kinsenoside의 확인, 순도, 함량시험을 위한 분석법을 개발하고, 이를 통하여 kinsenoside의 용매, 혈장, 간마이크로좀에서의 안정성을 평가하였다. Kinsenoside의 대사 및 약동학 연구를 위하여 생체시료에서의 LC/MS/MS 분석법 개발 및 validation을 수행하였으며, 개발된 분석법을 기반으로 rat에서의 혈장 약물 농도를 분석하고, 약동력학

본 연구에서는 고지혈증 치료제 개발을 위한 cholesterol ester transfer protein (CETP) 차단제 후보물질의 전임상연구를 수행하고자, kinsenoside의 확인, 순도, 함량시험을 위한 분석법을 개발하고, 이를 통하여 kinsenoside의 용매, 혈장, 간마이크로좀에서의 안정성을 평가하였다. Kinsenoside의 대사 및 약동학 연구를 위하여 생체시료에서의 LC/MS/MS 분석법 개발 및 validation을 수행하였으며, 개발된 분석법을 기반으로 rat에서의 혈장 약물 농도를 분석하고, 약동력학 특성을 규명하였다. 또한 kinsenoside의 약물상호작용을 평가하고자, CYP 효소에 대한 억제 작용을 평가하였으며, 고혈압치료제 및 타기전의 고지혈증 치료제와의 병용투여에 대한 약물상호작용을 평가하였다.

(출처 : 요약서 3p)

Abstract ▼

Kinsenoside (3-(R)-3-β-D-glucopyranosyloxybutanolide), is the principal bioactive constituent of Anoectochilus formosanus, an important ethnomedicinal plant in Asian countries, exhibited a variety of pharmacological actions. Kinsenoside was selected as a new drug candidate for hyperlipidemia with es

Kinsenoside (3-(R)-3-β-D-glucopyranosyloxybutanolide), is the principal bioactive constituent of Anoectochilus formosanus, an important ethnomedicinal plant in Asian countries, exhibited a variety of pharmacological actions. Kinsenoside was selected as a new drug candidate for hyperlipidemia with ester transfer protein (CETP) inhibitory effects. In this study, the preclinical efficacy testing of kinsenoside was conducted. The detailed research methodologies and the results are as follows.

The study started from the determination of kinsenoside by HPLC with UV detection, and found a very poor peak results as its wavelength range is below 200 nm. So we change from UV to ELSD (evaporative light scattering detector) for kinsenoside proper and selective detection, and obtained the satisfactory results. The purity of kinsenoside was determined by an HPLC-ELSD. The resulting data showed a purity of >98%. For more accuracy and selectivity, we proceed our investigation with mass spectrometry, the popular method among others. A reversed-phase LC-MS method for kinsenoside was developed by using waters UPLC system. Mass detection was performed in positive as well as negative ion mode, showed a better result in positive ion mode. For multiple reaction monitoring (MRM) analysis, the precursor-product ion pair used was 265.1→163.0 and 265.1→102.9 based on the product ion spectrum of kinsenoside. By this LC-MS method, we evaluated kinsenoside stability in solvent (chemical), plasma, and human/rat liver micosomes. Results showed that kinsenoside was well stable in the solvent medium as well as human and rat liver microsomes. We also observed that kinsenoside was highly unstable in rat plasma (ex vivo) at room temperature and at a neutral or basic pH. When it was incubated in plasma at an unmodified physiological pH (pH ≥7) and ambient temperature, kinsenoside showed ~50% degradation after 30 min and almost complete degradation (>90%) after 2 h.

Then the study was conducted for the improvement of kinsenoside plasma stability, as well as its column retention, by using HILIC system, as kinsenoside is highly hydrophilic and polar, so has poor retention in reverse phase column (previously studied). This was a big hurdle for us, to improve kinsenoside plasma stability, as it is highly unstable in plasma (as discussed in the previous study), and with HILIC application and optimization. We troubleshoot the expected stability problem, probably it's ex vivo interconversion (between lactone and hydroxyl carboxylic acid forms) and also hydrolysis by plasma esterases, had to be reduced by acidifying and enzymatic/nonenzymatic degradation of plasma. We improved the ex vivo stability of kinsenoside by controlling the pH (pH ~4.5 with acetic acid), temperature (the sample was processed on ice at 0-4° C and centrifuged at 4° C), and time (the collected plasma samples were readily acidified). In mass spectrometry, mass detection was performed in positive ion mode, and for selected reaction monitoring analyses, the precursor-product ion pairs used were m/z 265.2 → m/z 102.9 for kinsenoside. So, finally we have developed an optimized and validated HILIC-MS/MS method for determination of kinsenoside. The developed method was applied to a pharmacokinetic study of kinsenoside after i.v. administration, and was rapidly eliminated in a biphasic fashion. The distribution volume of kinsenoside was higher than the volume of body water, indicating that kinsenoside was well distributed in the tissues. In this report, for the first time we demonstrated the plasma pharmacokinetic properties of kinsenoside.

The effects of kinsenoside on CYP enzyme-mediated drug metabolism was evaluated in order to predict the potential for kinsenoside-drug interactions. Kinsenoside was tested at different concentrations of 0.1, 0.3, 1, 3, and 10 μM in human liver microsomes. The cocktail probe assay based on liquid chromatography-tandem mass spectrometry was conducted to measure the CYP inhibitory effect of kinsenoside. Subsequently, the metabolism profiles of amlodipine and lovastatin in human liver microsomes were analyzed following co-incubation with kinsenoside. The concentration levels of the parent drug and the major metabolites were compared with the kinsenoside-cotreated samples. The effect of kinsenoside was negligible on all CYP isozymes activities at the tested concentrations. The drug-drug interaction assay also showed that the concomitant use of kinsenoside has no effect on the concentration of amlodipine or lovastatin, and their major metabolites. So, it was concluded that there is almost no risk of drug interaction between kinsenoside and CYP drug substrates via CYP inhibition.

The resulting data shows that kinsenoside exhibits good efficacy as well as good pharmacokinetic profile and safety. Thus, it is expected that IND application on kinsenoside would be successfully processed. To optimize the new drug candidate, additional studies on the characterization of the active metabolites of kinsenoside and preclinical efficacy and pharmacokinetic evaluation with kinsenoside-related derivatives will be further conducted. In addition, based on the network between Korea and China research teams, further study on the development of bioactive natural product compounds will be carried out.

(출처 : SUMMARY 6p)

목차 Contents

• 표지 ... 1
• 제 출 문 ... 2
• 보고서 요약서 ... 3
• 요 약 문 ... 4
• SUMMARY ... 6
• CONTENTS ... 8
• 목차 ... 9
• 제1장 연구개발과제의 개요 ... 10
• 1. 연구개발의 필요성 및 배경 ... 10
• 2. 연구개발의 목표 및 범위 ... 11
• 제2장 국내외 기술개발 현황 ... 13
• 1. 국내외 관련분야의 기술개발현황 ... 13
• 2. 본 연구의 가치 및 중요성 ... 16
• 제3장 연구개발 수행내용 및 결과 ... 17
• 1. 연구개발 내용 및 방법 ... 17
• 2. 연구개발 결과 ... 26
• 3. 중국 연구팀과의 공동 연구 ... 37
• 제4장 목표달성도 및 관련분야에의 기여도 ... 42
• 1. 연구개발목표의 달성도 및 자체평가 ... 42
• 2. 관련분야에의 기여도 ... 44
• 3. 연구 성과 ... 45
• 제5장 연구개발결과의 활용계획 ... 47
• 1. 추가연구의 필요성 ... 47
• 2. 타 연구에의 응용 ... 47
• 제6장 연구개발과정에서 수집한 해외과학기술정보 ... 48
• 제7장 참고문헌 ... 50
• 끝페이지 ... 54