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Functional Studies on Genetic Variations in Human Organic Anion Transporting Polypeptide 1B Transporters : 유기 음이온 수송 폴리펩타이드 1B 수송체의 유전적 변이형에 관한 기능 연구

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Authors
채윤지
Advisor
정석재
Major
제약학과
Issue Date
2012-02
Publisher
서울대학교 대학원
Abstract
PART I. Functional Consequences of Genetic Variations in the Human Organic Anion Transporting Polypeptide 1B3 (OATP1B3) in the Korean Population
The objectives of this study were to investigate the allele frequencies and linkage disequilibrium (LD) in the organic anion transporting polypeptide 1B3 (OATP1B3) in the Korean population and to examine the functional consequences. Using samples from 48 Koreans, direct sequencing was carried out to determine the allele frequencies and LD of OATP1B3 in a representative Korean population. Thirty-six genetic variations in the transporter were found in Koreans; among them, five undocumented variations (i.e.,−6436G>C in the 5-upstream region, 26A>C and 586A>G in the protein coding region, and IVS6-72A>T and IVS12-80A>T in intron regions) were identified. In the upstream region, −5035G>A was found to have lowered gene expression, as determined by a reporter gene assay, suggesting that this variation reduces the expression of OATP1B3 in humans. The functional relevance of the genetic variations in the protein coding region was determined by an uptake study involving representative substrates in human embryonic kidney 293 cells expressing wild type or variant forms. Variations involving 699G>A showed a reduced uptake activity for testosteron but not for estradiol 17β-D-glucuronide or methotrexate, indicating that the functional impact of the variations is substrate specific. Considering the kinetic relevance of OATP1B3, the functionally affected variations may be therapeutically important.

PART II. In vivo Expression of Human Organic Anion Transporting Polypeptide 1B1 (hOATP1B1) and its Genetic Variant 521T>C in the Mouse Liver
The objective of this study was to examine the feasibility of in vivo expression of human organic anion transporting polypeptide 1B1 (hOATP1B1) forms in the mouse liver by hydrodynamic gene delivery for the application in the pharmacokinetic study of the genetic variation. Alterations in serum biochemistry and hepatocyte histology, rendered by hydrodynamic gene delivery (HGD), were apparently transient and reversible. The expression of a reporter gene was found to be liver-specific; among the gene delivery vectors, pCMV showed the highest level of expression, as determined by the reporter gene expression, in the liver. The reporter gene expression was detected in the plasma but not in the blood cell in mice subjected to HGD, probably due to transiently increased permeability of hepatocytes by HGD and subsequent release of the protein from the hepatocyte to the systemic circulation. Under this experimental condition, the expression of hOATP1B1 was readily detected in the mouse liver, but not in other tissues, receiving injection of the plasmid containing the gene. Compared with the sham control mice, the uptake of pravastatin increased significantly in mice expressing hOATO1B1 wild type; the uptake decreased in mice expressing the 521T>C variant. These observations suggest that the functional expression of human transporter gene in mice is feasible and that the model appears adequate for in vivo pharmacokinetic studies. Particularly, this experimental tool may be useful in the study of genetic variation of hOATP1B1 in vivo prior to clinical study.
Language
eng
URI
https://hdl.handle.net/10371/156648

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College of Pharmacy (약학대학)Dept. of Manufacturing Pharmacy (제약학과)Theses (Ph.D. / Sc.D._제약학과)
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