Conclusions: CETA may be a more sensitive system than the bi-directional transport assay to detect transport of drugs with high passive diffusion across the BBB. We conclude that PHT, PB, OXC, ESL, CBZ-E, S-LC, and LCM, but not ESM, CBZ, RPM, ZNS, and PGB, are transported by human Pgp. These data suggest that resistance to PHT, PB, ESL, OXC and LCM might be attributed to increased efflux function of Pgp because they or their active metabolites are Pgp substrates. The CTC haplotype exhibited increased directional transport activity by Pgp. The effects of MDR1 polymorphisms on AED transport may provide a molecular explanation of the association between the polymorphisms and pharmacoresistance. This knowledge may help guide the design of genetic-based individualized therapy of epilepsy. / Methods: Stable transfected clones of human MDR1 haplotypes combining 1236C>T, 2677G>T/A, and 3435C>T in LLC-PK1 cells were established and validated. The expression level and localization of Pgp were measured. Bi-directional transport assays or concentration equilibrium transport assays (CETA) were performed by using MDR1-transfected and non-transfected cells to determine the substrate status of the following AEDs: phenytoin (PHT), phenobarbital (PB), ethosuximide (ESM), carbamazepine (CBZ), eslicarbazepine acetate (ESL), oxcarbazepine (OXC), (S)-licarbazepine (S-LC), carbamazepine-10,11-epoxide (CBZ-E), rufinamide (RFM), lacosamide (LCM), zonisamide (ZNS), and pregabalin (PGB). LLC-PK1 cells transfected with MDR1 variants were used to evaluate the effects of MDR1 polymorphisms on transport activity of AEDs in CETA. / Purpose: Epilepsy is a major neurological disorder, affecting more than 50 million people worldwide. Antiepileptic drugs (AEDs) do not effectively treat 30--40% of patients. Export of AEDs by P-glycoprotein (Pgp, ABCB1, or MDR1), which is overexpressed in the blood-brain barrier in drug-resistant patients, may be a mechanism for resistance to AEDs. Single nucleotide polymorphisms (SNPs) 1236C>T, 2677G>T and 3435C>T have been associated with drug-resistant epilepsy and were sometimes found to have effects on Pgp activities. But whether (or which) AEDs are transported by Pgp remains unclear, and there is no direct evidence showing that polymorphisms affect the transport of AEDs by Pgp. Therefore, we propose to use monolayers of cells transfected with the MDR1 variants to investigate 1) which AEDs are substrates for Pgp; and 2) the effect of MDR1 polymorphisms (1236C>T, 2677G>T, and 3435C>T) on AED transport. / Results: In CETA, PHT, PB, and LCM were transported by MDR1-transfected cells from basolateral to apical sides, while RFM, ZNS, PGB and ESM were not transported. Pgp did not transport CBZ, but did transport its active metabolite CBZ-E. Pgp also pumped ESL, OXC, and their active metabolite S-LC. The transport of these drugs can be completely blocked by Pgp inhibitor verapamil or tariquidar. In bi-directional transport assays, the Papp for the basolateral to apical direction in MDR1-transfected cells was significantly higher than in non-transfected cells for PHT, OXC, ESL, and S-LC, and not for PB, CBZ-E, CBZ, or ESM. / To compare the extent of basolateral-to-apical transport efficiency of different variants, we calculated the amount of the transported drugs divided by expression level of MDR1 in the apical chamber for each variant. In the G418 selection condition, compared with reference haplotype CGC, the CTC haplotype increased Pgp activity to transport OXC and ESL, while the CGT and CTT haplotypes did not significantly affect Pgp function. In the vincristine sulfate selection condition, compared with CGC, the haplotype CTT decreased Pgp activity, while other haplotypes, including CGC, CGT, CAC, CTC, TGC, TGT, TTT, and TTC, did not affect function. Selection by vincristine sulfate may raise expression of Pgp and eliminate differences among the variants. / Zhang, Chunbo. / Advisers: Vincent Hon Leung Lee; Lawrence William Baum; Zhong Zuo; Patrick Kwok Leung Kwan. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 192-221). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_344987 |
| Date | January 2011 |
| Contributors | Zhang, Chunbo, Chinese University of Hong Kong Graduate School. Division of Pharmacy. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, theses |
| Format | electronic resource, microform, microfiche, 1 online resource (xx, 221 leaves : ill. (some col.)) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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