No description available.

A epilepsia, doença descrita pela primeira vez em 2000 a.C., tem a crise convulsiva ou epiléptica como fenômeno paroxístico, e a International League Against Epilepsy define a crise epiléptica como \"manifestação excessiva e/ou hipersincrônica, normalmente autolimitada, da atividade dos neurônios no cérebro\". Há 40 anos surgiram os medicamentos antiepilépticos, mas a resistência múltipla a fármacos antiepilépticos (FAEs) é um problema significante que afeta pelo menos 30% dos pacientes portadores dessa doença devastadora. O mecanismo exato da fármaco-resistência desenvolvida em pacientes epilépticos ainda é desconhecido, porém uma possível causa seria a inadequada acumulação intraparenquimal do fármaco antiepiléptico relacionada a expressão aumentada da glicoproteína-P (PgP). Neste contexto, nosso objetivo foi investigar a correlação da expressão da PgP, codificada pelo gene ABCB1, no córtex temporal de 12 pacientes fármaco-resistentes frente aos FAEs fenobarbital, carbamazepina, fenitoína e lamotrigina; comparamos também a expressão da PgP nesse mesmo grupo de pacientes, selecionados no Centro de Cirurgia de Epilepsia (CIREP) frente a um grupo controle composto por indivíduos não epilépticos que evoluíram para óbito examinados no Serviço de Verificação de Óbito do interior (SVOi). Utilizamos a Cromatografia Líquida de Alta Eficiência para determinar as concentrações dos FAEs no plasma e no cérebro, sendo que a metodologia utilizada no tecido cerebral foi desenvolvida e validada especialmente para esse fim. Essas concentrações foram então determinadas e a razão entre as duas medidas foi comparada com a expressão de PgP no tecido cerebral. Analisando os resultados concluímos que não há correlação linear entre a razão dos fármacos estudados e a expressão de PgP no córtex temporal de pacientes com epilepsia refratária. / Epilepsy, desease described for the first time in 2000 B.C., presents the convulsion or epileptic seizure as its paroxysmal event, and the International League Against Epilepsy defines the epileptic seizure as \"excessive and/or hypersynchronous manifestation, usually self-limiting, from brain neurons activity\". Forty years ago the antiepileptic drugs (AEDs) emerged, but multiple resistance to AEDs is a significant problem which affects at least 30% of epileptic patients. The mechanism underlaying pharmacoresistance is still unknown, but a possible cause is the inadequate accumulation of AEDs in the brain tissue related to P-glycoprotein over expression. In this context, our aim was to investigate the correlation between PgP expression, codified by ABCB1 gene, in temporal cortex of 12 pharmacoresistant patients towards AEDs phenobarbital, carbamazepine, phenytoin and lamotrigine; we also compared PgP expression between the same group of patients, selected inside Epilepsy Surgery Center (CIREP), and a control group composed by non-epileptic deceased individuals examined at the Death Verification Service from our Medicine School (SVOi). We used High Performance Liquid Chromatography to determine AEDs concentrations in plasma and brain, and the methodology applied to brain quantification was specially developed for this purpose. These two concentrations were then determined and the ratio between them was compared with PgP expression in brain tissue. Analyzing the results we concluded that there is no linear correlation between the ratio of the AEDs studied and PgP expression in temporal cortex of patients with pharmacoresistant epilepsy.

Schweinfurthins are intriguing natural product chemotherapeutics due to their potent yet selective activity and their unknown mechanism of growth inhibition in cancer. Much progress has been made in characterizing the intracellular effects of the schweinfurthins since they were first isolated from Macaranga schweinfurthii in 1986. Here, the L-type calcium channel and P- glycoprotein (Pgp) inhibitor verapamil has been found to enhance schweinfurthin- induced growth inhibition. Verapamil induces an increase in the intracellular concentration of a fluorescent schweinfurthin. However, the synergistic relationship between the schweinfurthins and verapamil is complex and not obvious in that verapamil fails to increase the intracellular concentration of a schweinfurthin analogue that is a known substrate of Pgp. Schweinfurthins are also found to induce alterations to cholesterol homeostasis by increasing the expression of the cholesterol efflux pump ABCA1 in an apparent liver X receptor- independent fashion. In addition, schweinfurthin treatment blunts epidermal growth factor downstream activation and phosphorylation of Akt. Lastly, a schweinfurthin-resistant cell line has been created and characterized for resistance to schweinfurthin-induced growth inhibition. The variety of intracellular effects characteristic of schweinfurthin treatment described here provide mechanistic framework for identifying the potential target and mechanism of growth inhibition for the schweinfurthins.

The gastrointestinal parasite, Haemonchus contortus, is of major concern in the sheep and goat industry as well as in zoological settings. Over the years this parasite has developed resistance to the three classes of anthelmintics, benzimidazoles, imidazothiazoles and macrocyclic lactones, that are currently used for treatment. One of the mechanisms proposed to be involved in this resistance is the efflux transporter P-glycoprotein (Pgp). In this study, the resistance status of several strains of H. contortus was evaluated using the larval development assay DrenchRite®. After documenting the resistance status of these strains, transcription of Pgp in L3 larvae after exposure to anthelmintics was quantitated using polymerase chain reaction (PCR). Of the strains analyzed, only one was determined to be susceptible to all of the anthelmintics tested, while the others showed variable levels of resistance to one or more. A Haemonchus strain acquired from a giraffe at a zoo in Florida was the most resistant, showing extremely high levels of resistance to benzimidazoles and levamisole. Molecular characterization of the 18S rRNA gene and the internal transcriber spacer region (ITS) were performed on the giraffe strain to identify the species. Although there were variations in the isolate sequences, the most likely species for the giraffe strain was H. contortus. No transcription of Pgp was identified in H. contortus L3 larvae under the conditions of this study. Thus, increased Pgp does not appear to be a primary mechanism of drug resistance in this stage of the worm.

Our laboratory has previously demonstrated that experimental peripheral inflammatory pain (PIP), induced by subcutaneous plantar injection of λ-carrageenan in Sprague Dawley rats, results in increased expression and activity of the ATP-dependent efflux transporter P-glycoprotein (P-gp) that is endogenously expressed at the blood-brain barrier (BBB). Increased P-gp functional expression was associated with a significant reduction in CNS uptake of morphine and, subsequently, reduced morphine analgesic efficacy. The present study examined whether the PIP-induced increase in P-gp functional expression was due to changes in intracellular trafficking (i.e., localization of P-gp), mediated by changes in the association of P-gp and caveolin-1, a key trafficking protein. These studies also determined if the drug diclofenac, a non-steroidal antiinflammatory (NSAID) that is commonly administered in conjunction with opioids during peripheral inflammatory pain (PIP), altered or modulated P-gp functional expression providing evidence of a drug-drug interaction. Confocal microscopy and subcellular fractionation revealed that under conditions of PIP, the disassembly of high-molecular weight P-gp-containing structures result in an increase in P-gp ATPase activity and changes in the localization of P-gp. Western blot analysis demonstrated further an increase in P-gp expression in rat brain microvessels following PIP induction and also after diclofenac treatment alone in the absence of PIP. Additionally, in situ brain perfusion studies showed that both PIP and diclofenac treatment alone increased P-gp efflux activity resulting in decreased radiolabeled- morphine uptake into the brain. This concurrent administration of NSAIDs and opioids in the presence of a pathophysiological stressor (i.e., pain/inflammation) may result in clinically significant drug-drug interactions that may impair the desired pharmacologic response and analgesic effects of opioids. Such interactions can lead to significant modifications to pain management in clinical settings. Therefore: The central hypothesis of this work is that the pathophysiological stressor peripheral inflammatory pain (PIP) and the pharmacological agent diclofenac modulate P-glycoprotein functional expression at the BBB. This hypothesis may be broken down further into two parts: 1) PIP induced changes in P-gp functional expression are mediated via changes in Pgp intracellular trafficking. 2) The non-steroidal anti-inflammatory drug Diclofenac, a drug commonly used to treat pain, modulates P-gp functional expression at the BBB thus decreasing morphine uptake into the CNS.

The ABC protein P-glycoprotein (Pgp, ABCB1) transports many structurally diverse substrates from the lipid bilayer. Previous studies demonstrated the importance of the membrane environment, but few have quantified these effects. In the present work, purified Pgp reconstituted into defined lipid systems was employed. Drug binding affinities were determined using Trp quenching, and drug-lipid partitioning by equilibrium dialysis. Pgp bound substrates from the bilayer with affinities in the millimolar range; both drug-Pgp and drug-lipid interactions were important. The kinetics of Pgp-mediated drug transport were sensitive to drug structure and lipid environment. The rate of transport is proposed to depend on the affinity of Pgp for substrate and conformational changes. The lipid bilayer affected the stability of Pgp catalytic activity which provided evidence for distinct basal and drug-stimulated ATPase cycles. Overall, the lipid environment had pronounced effects on Pgp-mediated drug binding, transport and catalytic functions. / Canadian Cancer Society

Anthelmintic treatment of livestock is an important aspect of the control of gastrointestinal parasites. Resistance to anthelmintics is common, and an understanding of resistance requires knowledge of an anthelmintic's mode(s) of action and mechanism(s) of resistance. The parasitic nematode, Haemonchus contortus, has developed resistance to benzimidazoles and avermectins/milbemycins. Proposed mechanisms of resistance are here supported by genetic changes observed in genes whose protein products are believed to interact with these anthelmintics. Statistically significant differences in allele frequencies were observed between untreated and ivermectin- and moxidectin-treated strains in a gene encoding a putative glutamate-gated chloride channel alpha subunit, a proposed target of avermectins/milbemycins. One allele appeared to be associated with resistance. Similar changes in allele frequencies in the same strains occurred in a gene encoding a subunit of a gamma-aminobutyric acid receptor. Significant differences in allele frequencies of a gene encoding a P-glycoprotein were found in strains of H. contortus treated with ivermectin and moxidectin compared to derived, untreated strains. In all treated strains, one allele appeared to be associated with resistance. Similarly, allele frequencies of this gene were significantly different between a cambendazole-treated strain and its derived, untreated strain. These results implicate glutamate-gated chloride channels and gamma-aminobutyric acid receptors in mechanisms of resistance to avermectins/milbemycins and implicate P-glycoprotein in a mechanism of resistance to avermectins/milbemycins and benzimidazoles in H. contortus.

Resistance to the avermectin/milbemycin class of anthelmintics in nematodes has become a serious problem worldwide due to their unrestricted usage. Resistance to these compounds is attributed to the over-expression of the transport protein, P-glycoprotein (P-gp). P-gp acts by pumping drug molecules out from the cell or organism, P-gp efflux activity can be blocked using multidrug resistance (MDR) modulators associated with chemotherapy to enhance their therapeutic effect. A series of experiments was undertaken to determine if the association of the anthelmintics, ivermectin (IVM) and moxidectin (MOX), and MDR modulators would increase the anthelmintics' efficacy against resistant parasites. Using an in vitro migration assay, IVM and MOX in the presence or absence of verapamil (VRP), CL347,099 and cyclosporin A (CyA) were used against IVM- and MOX-selected strains of H. contortus. The modulators alone had no effect on reducing the number of migrating larvae, IVM and MOX had a significant increase in efficacy of 52.7 and 58,3% respectively, when used in association with VRP, above that obtained with the anthelmintics alone. CL347,099 was also able to significantly increase the IVM and MOX efficacy by 24.2 and 38.9%, respectively. The effect of IVM and MOX in combination with VRP and CL347,099 was determined in jirds infected with selected strains of H. contortus. The combinations of VRP with either IVM or MOX significantly reduced worm counts of the selected strains compared with the untreated controls, whereas IVM or MOX alone did not. CL347,099 plus MOX combination was significantly more efficacious than moxidectin alone against the selected strains. To evaluate the effect of VRP on the pharmacokinetic behaviour of the anthelmintics IVM and MOX, the drug combination was given to sheep. The IVM plus VRP treatment resulted in an increase of the pharmacokinetic parameters of IVM. The peak concentration (83%) and area under the curve (54%) were significantly differen

Anthelmintics, antiparasitic agents, have been developed as a main weapon to control parasitic nematodes of domestic ruminants. Unfortunately, the intensive use of anthelmintics leads to the development of drug resistance in parasite populations. Anthelmintic resistance has compromised the control of nematode parasites and has become a major problem in many countries of the world. Resistance to the newest anthelmintics such as ivermectin (IVM) and related anthelmintics in Haemonchus contortus in sheep has been developing rapidly in recent years. The development of drug resistance is an evolutionary process that leads to genetic changes in parasite populations in response to drug exposure. However, the mechanism of ivermectin resistance in nematode parasites is unknown. P-glycoprotein (Pgp) has been well documented in mammalian cells as a membrane transporter by actively extruding a variety of structurally and functionally unrelated hydrophobic cytotoxic drugs out of the cell. This study was to determine whether there is an association between specific alleles at the Pgp locus and IVM or moxidectin (MOX) selection in H. contortus, by investigating the genetic variation of the Pgp homologue in unselected and IVM- and MOX-selected strains of H. contortus. (Abstract shortened by UMI.)

Little is known about potential influences of kinase pathway modulation on expression and activity of P-glycoprotein (P-gp). A protein kinase inhibitor (PKI) library was screened, to determine its effects on activity and expression of P-gp, in various cell lines. Cell lines were incubated with PKI for 24 h. Subsequent P-gp substrate accumulation studies were performed. Changes in P-gp activity and/or expression ≥ 25% compared to control were considered hits. Kinase pathways identified as P-gp activity hits were examined for their ability to modulate permeability. PKI families GSK-3, Craf1 and VEGFR2 and Tie-2, significantly modulated P-gp activity in the MDCK cell line. PKI families GSK-3, Iκκ and Jnk2/3 significantly modulated P-gp activity in the Caco-2 cell line. Few P-gp activity hits significantly modulated P-gp expression. PKIs modulate P-gp activity more than P-gp expression in a cell line dependent manner, excluding GSK-3 PKI family, which appears to be cell line independent.