Abstract In previous chlorpromazine pharmacokinetic studies a dramatic elevation in blood plasma levels of this drug was observed when taken in combination with oral contraceptives. Different mechanisms have been postulated to explain this observation. The aim of the study was to investigate whether oral contraceptives such as ethinyloestradiol and progesterone enhance the absorption of chlorpromazine by means of inhibiting P-glycoprotein (P-gp) and if this effect is mainly due to ethinyloestradiol or progesterone or their combination. The Caco-2 cell line was used as an in vitro model to study the effects of these compounds on the transport of chlorpromazine. Both apical to basolateral (AP-BL) and basolateral to apical (BL-AP) transport studies were done on chlorpromazine in combination with different compounds. Ethinyloestradiol enhanced the AP-BL cumulative transport of chlorpromazine by 11.5% compared to the control group, which was also statistically significantly higher than the effect caused by progesterone (0.8%). A combination of these two steroidal hormones enhanced the cumulative transport of chlorpromazine by only 2.0% compared to the control group. This indicates the possible existence of separate drugbinding sites for these two hormones and chlorpromazine on P-gp. The drug-binding site (or receptor) for progesterone probably interacts allosterically with the binding site for ethinyloestradiol and thereby decreasing its transport enhancing effects on chlorpromazine.

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.

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Thesis (Ph. D.)--Massachusetts Institute of Technology and Woods Hole Oceanographic Institution, 2000. / "February, 2001." "Funding was provided by National Institute of Health grant ES-07381 and a Rinehart Coastal Research Center grant." Includes bibliographical references (p. 174-177).

P-glycoprotein (Pgp; ABCB1) is a well known transporter involved in energy-dependent-drug efflux activity. At the brain capillary endothelium, its luminal membrane location is ideal for its ascribed role in the physiological efflux of a wide array of structurally and functionally diverse compounds from the brain. This is a critical issue in regards to the delivery of central nervous system (CNS)-acting therapeutics. Moreover, a dysregulation of Pgp has been implicated in specific CNS disease states, including Alzheimer's disease, epilepsy, and brain cancer where an upregulation of Pgp has been well established as a mediator of multi-drug resistance. Inflammation is a common component in all of these conditions. Previously our laboratory has reported changes in BBB molecular and functional properties during inflammatory pain (Huber et al. 2001). This has led us to investigate the effects of peripheral inflammatory pain on Pgp efflux transport properties at the BBB, in vivo. In the present study we examined the effects of lambda-carrageenan-induced inflammatory pain (i.e. hyperalgesia; CIP) on the molecular and functional properties of Pgp at the BBB. Western blots using enriched fractions of isolated rat brain microvessels revealed that Pgp expression at the BBB was increased by CIP and that this increase occurred predominantly within the membrane region of the cell. Additionally, both in situ brain perfusions and whole body antinociceptive profiling of the Pgp substrate and opioid analgesic, [3H] morphine, indicate that changes in Pgp at the BBB, mediated by peripheral inflammation, can impact brain uptake of morphine. To further elucidate the mechanism(s) behind the rapid upregulation (3 h) of Pgp at this region, we explored regulation of Pgp at the plasma membrane. Our findings show that CIP induces a movement of Pgp within these domains and that Pgp co-localizes with caveolin-1 and clathrin, key structural proteins associated with caveolae and clathrin-pit lipid rafts, respectively. Our data indicate for the first time that peripheral inflammatory pain induces functional and molecular changes in Pgp, a critical efflux transporter, at the BBB in vivo and that these alterations may be mediated in part via a proteolipidic re-organization mechanism.

The development of multidrug resistance (MDR) in tumour cells to a wide range of anticancer drugs has become a major obstacle in the chemotherapeutic treatment of cancer. Molecular characterization of MDR tumour cells has led to the identification of several cell-based genetic alterations including the overexpression of a membrane protein, P-glycoprotein (P-gp). P-gp is a ATP dependent drug efflux pump and P-gp ATPase activity has been demonstrated to be essential in drug transport. In an effort to understand how P-gp ATPase activity is coupled to drug binding and transport, we examined the effects of N-ethylmaleimide (NEM), a potent inhibitor of P-gp ATPase, on P-gp drug binding and transport. Our results show that short term treatment of MDR cells with NEM led to a concentration-dependent increase in P-gp drug binding and phosphorylation. In addition, NEM increases [3H]-vinblastine accumulation in drug resistant cells but not in sensitive cells. Our study suggests that inhibition of P-gp ATPase activity, and not increased phosphorylation of P-gp by NEM, is responsible for the observed increase in P-gp-drug binding. / Selection of tumour cell lines in vitro has led to multiple cellular changes that may mediate drug resistance to anticancer drugs. The role of other mechanisms, in addition to P-gp and multidrug resistance protein (MRP) in drug resistance, is supported by evidence from studies with tumour cell lines and clinical tumours. In an effort to identify other cellular changes that may be important in tumour drug resistance to anticancer drugs, we have used a differential immunodot blot method to isolate monoclonal antibodies that bind to proteins in drug resistant but not in drug sensitive cells. By using the immunodot blot method, we have isolated a monoclonal antibody (IPM96) which recognized a 40 kDa protein (P-40) in several MDR cell lines. The expression of P-40 is concurrent with the level of drug resistance. Biochemical characterization showed P-40 to be associated with the cell membrane and in the soluble fraction. Molecular cloning of P40 cDNA revealed that P-40 is identical to annexin I, a substrate for the epidermal growth factor receptor tyrosine kinase. The observed increase in P-40 (or annexin I) protein levels in drug resistant cells is due to the elevation of P-40 transcripts. The pharmacological characterization of P-40 cDNA transfectants (P-40-MCF-7) has demonstrated that overexpression of P-40 in drug sensitive cells is capable of conferring drug resistance to adriamycin, actinomycin D, Taxol and cisplatin. Taken together, our study provides convincing evidence that annexin I is important in the development of drug resistance in cancer cells. In addition, it suggests a novel mechanism of drug resistance that is different from the ATP-dependent drug efflux pumps that mediate P-gp- and MRP-associated MDR

Resistance to ivermectin (IVM) in Haemonchus contortus has developed in many countries and its mechanism is still under investigation. P-glycoproteins (P-gp) are transmembrane proteins that can transport drugs out of cells. Researchers have found that there is polymorphism in a P-gp gene from H. contortus between IVM-selected and unselected worms. Three main P-gp polymorphs were identified, polymorph A was found to be related to IVM selection, while polymorphs B and X were associated with susceptibility. The purpose of this research is to investigate the genetic variations in P-glycoprotein that are associated with IVM selection or susceptibility in H. contortus. Total RNA and genomic DNA were extracted from individual male adult worms of IVM-selected and unselected strains of H. contortus. A fragment of the P-gp gene was amplified from the genomic DNA of individual worms and RFLP analysis was performed on the PCR product to genotype the corresponding worms. The homozygous worms that possessed polymorph A, B or X were identified.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser.

Abstract Apoptosis is a biochemically regulated mechanism leading to the destruction of an individual cell. An inadequate apoptosis is partly responsible for uncontrolled survival of malignantly transformed cells and formation of cancer. The growth of a tumor depends on the proliferative capacity and destruction of tumor cells either through apoptosis or necrosis. In this work, the extent of apoptosis and the expression of apoptosis-regulating proteins were studied by 3'-end labeling of fragmented DNA (TUNEL) and immunohistochemistry in a set of 166 tissue samples consisting of 33 HCCs, 39 gallbladder carcinomas, 7 gallbladder dysplasias and 87 pancreatic carcinomas. In addition, p53 protein and P-glycoprotein expression was studied immunohistochemically. The extent of apoptosis was estimated by using apoptotic index, defined as a percentage of apoptotic cells in the entire tumor cell population. The present results show an average apoptotic index of 0.73% in HCC, 0.68% in gallbladder and 0.69% in pancreatic carcinoma. Bcl-2 positivity was found in only 3% of the HCCs, 10% of gallbladder and 13% of pancreatic carcinomas. Bax positivity was seen in all of the gallbladder and pancreatic carcinoma cases. Mcl-1 positivity was found in 87% of gallbladder and 86% of pancreatic tumors. The apoptotic index in bcl-2 positive cases was lower (0.35%) than in cases showing no immunoreactivity (0.64%) in pancreatic tumors (P = 0.013). Apoptotic index was higher in pancreatic tumors with strong bax immunoreactivity (0.70%) than in other cases (0.34%) (P = 0.002). Caspase 3, 6 and 8 expression was found in 92%, 92% and 73% of HCC, 95%, 77% and 77% of gallbladder carcinoma and 80%, 80% and 74% of pancreatic carcinoma cases, respectively. p53 positivity was found in 23% of hepatocellular, 57% of gallbladder and 41% of pancreatic carcinomas. P-glycoprotein was observed in 65% of the HCCs. Patients with Pgp positive tumors had a significantly shorter disease-free interval than those with Pgp negative tumors (P < 0.05). To evaluate the growth potential of HCC and pancreatic carcinoma, a growth index from the scores obtained for apoptosis, necrosis and cell proliferation was designed. Patients with a high degree of proliferation relative to the degree of necrosis and apoptosis (i.e. had a positive growth index) in HCC lesions had a significantly shorter survival (P = 0.004) and disease-free interval after operation (P = 0.019) than those with a tumor predominated by apoptosis and necrosis. Results were in line with HCC in pancreatic carcinoma, but the association did not reach statistical significance (P = 0.09). According to the results the extent of apoptosis was similar in HCCs, gallbladder and pancreatic carcinomas. These tumors also showed here a similar expression pattern of the bcl-2 family of proteins and caspases. None of the individual parameters associated significantly with apoptosis except for bcl-2 and bax in pancreatic carcinoma, neither was there any association between p53 and P-glycoprotein expression and apoptosis. Calculation of a growth index might be helpful in assessing the prognosis of patients with tumors with a scant stroma, such as HCC.

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