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Study on Factors Limiting Intestinal Drug Absorption: Contribution of interplay between metabolism and efflux transport in intestinal epithelial cells, and intestinal stability on the absorption

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Authors
유헌민
Advisor
정석재
Major
약학대학 약학과
Issue Date
2017-02
Publisher
서울대학교 대학원
Keywords
Intestinal P-glycoproteinIntestinal first pass metabolismP-gp recyclingIndinavirHM-30181AEC-181-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerolPharmacokineticsLC-MS/MS
Description
학위논문 (박사)-- 서울대학교 대학원 : 약학과 약제과학 전공, 2017. 2. 정석재.
Abstract
PART I. Enhanced intestinal absorption of indinavir and its metabolic conversion to M6 by the co-administration of HM-30181A, a P-glycoprotein inhibitor

Intestinal P-glycoprotein (P-gp), an efflux transporter, may render its substrate to recycle during the intestinal absorption process. The objective of this study is to determine the contribution of P-gp mediated recycling in the intestinal first pass metabolism. To examine whether HM-30181A, a potentially specific inhibitor for P-gp, interacted with CYP enzymes (i.e., 1A2, 2C9, 2C19, 2D6, 3A4), the metabolic stability of standard substrates for major CYP enzymes in rat liver microsomes was studied in the presence and the absence HM-30181A. Furthermore, to determine whether compound interacted with typical SLC transporters (i.e., rOatp1b2, rOat1, rOat3 and rOct2), the uptake of their standard substrates in MDCKII/FRT cells expressing the transporters was studied with or without the compound. In this study, indinavir was selected as the model compound since the drug is a well-established substrate for both P-gp and CYP3A4. Rats orally received the dose of 10 mg/kg of HM-30181A, 10 minutes prior to the indinavir administration (10 mg/kg orally). For portal vein infusion study, rats received indinavir infusion via portal vein catheter at the rate of 15 mg/kg/h for 20 min while the inhibitor was given orally 10 min prior to the initiation of the infusion. It was found that HM-30181A did not affect the activity of typical CYP enzymes (1A2, 2C9, 2C19, 2D6 or 3A4) and SLC transporters (rOatp1b2, rOat1, rOat3, rOct2) suggesting that the compound was a specific inhibitor for P-gp. In oral administration study, Cmax values of indinavir (Cmax, IND) for control and HM30181-treated group were (in mean ± SD) 0.614 ± 0.083 and 1.51 ± 0.64 μg/mL, respectively. AUC values for indinavir (AUCIND) of the two groups were also affected similarly [viz, in HM-30181 treated group, AUC approximately 111% higher (p<0.05) than control group]. AUC of M6 (AUCM6) was increased in HM-30181A-treated group to the similar extent of the increase found with AUCIND. For the case of portal vein infusion study, Cmax, IND, AUCIND and AUCM6 were 1.4, 2.5 and 1.9-fold increased when HM-30181A was co-administered. In Ussing chamber study, Papp values were 2.85 ± 0.19 and 4.34 ± 1.08 X 10-6 cm/sec for untreated and HM-30181A treated group, respectively. Amount of indinavir was tend to increase (2650 ± 1130 and 3290 ± 1110 pmole/g tissue, respectively), although the values were not statistically different. For the case of M6 conversion, the amount of the metabolite was increased (54.9 ± 10.9 and 77.3 ± 16.3 pmole/g tissue, respectively, p < 0.05) in the intestine when the tissue was treated with HM-30181A, consistent with a simulation result with a kinetic model assuming cooperative action of recycling and metabolism. Therefore, the in vivo observations with different routes of indinavir administration, ex vivo study and in silico study collectively showed that the absorption and metabolism of indinavir is slightly enhanced by HM-30181A.

PART II. Quantification of EC-18, a synthetic monoacetyldiglyceride (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol), in rat and mouse plasma by liquid-chromatography/tandem mass spectrometry

EC-18 (i.e., 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol), an active ingredient in Rockpid®, has been reported to be useful in controlling various types of inflammations, particularly those caused by neutropenia. Although this product was originally approved as a functional food in Korea, it is currently in phase II clinical trials for use in managing the severe chemotherapy-induced neutropenia in patients with advanced breast cancer who are receiving intermediate febrile neutropenia risk chemotherapy. The objective of this study was to develop a rapid, sensitive method for the determination of EC-18 in rat and mouse plasma and to evaluate the applicability of the assay in pharmacokinetic studies. EC-18 was extracted with MeOH from rat and mouse plasma samples, and the extract directly introduced onto an LC-MS/MS system. The analyte and EC-18-d3, an internal standard, were analyzed by multiple reaction monitoring (MRM) at m/z transitions of 635.4→355.4 for EC-18 and 638.4→338.4 for the internal standard, respectively. The lower limit of quantification (LLOQ) was determined at 50 ng/mL, with an acceptable linearity in the range from 50 to 10000 ng/mL (r > 0.999) for both matrices. Validation parameters such as accuracy, precision, dilution, recovery, matrix effects and stability were found to be within the acceptance criteria of the assay validation guidelines, indicating that the assay is applicable for estimating EC-18 in concentrations in the range examined. EC-18 was readily determined in plasma samples for periods of up to 8 h following an intravenous bolus injection of 1 mg/kg in rats and at 5 mg/kg in mice, respectively, and up to 24 h following the oral administration of 2000 mg/kg in mice. The findings indicate that the current analytical method is applicable for pharmacokinetic studies of EC-18 in small animals.
Language
English
URI
https://hdl.handle.net/10371/120167
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College of Pharmacy (약학대학)Dept. of Pharmacy (약학과)Theses (Ph.D. / Sc.D._약학과)
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