Abstracts
The problem of drug resistant epilepsies
J. W. Sander
Institute of Neurology, University College London, Queen Square, London WC1 3BG, UK
For the majority of patients with epilepsy, the prognosis for seizure control is very good; however, refractory epilepsy develops in about 20–30% of patients and represents a significant challenge for both clinical management and for research. Physicians treating epilepsy are often asked to predict prognosis and make decisions about commencing and withdrawing treatment. Practice can be guided by epidemiological studies of prognosis. It seems that prognosis depends largely on the aetiology of the seizures and the clinical background of the patient rather than on the seizures themselves or the treatment prescribed. Aetiologies are, however, not the sole determinant of outcome and response to treatment and unknown factors must exit. It is likely that some of these unknown factors are genetically determinate but this needs confirmation. The search for these unknown factors that may determine intractability in epilepsy is a very exciting prospect, which may prove to be multi-factorial. In some patients, for instance, epilepsy may indeed become resistant to treatment whilst in others the propensity for intractability to the drugs currently available may be part and parcel of the condition. Further research is urgently needed to elucidate the full range of mechanisms that lead to drug resistant epilepsy.
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©2002 The Novartis Foundation
Drug resistance molecules: lessons from oncology
George L. Scheffer and Rik J. Sheper
Department of Pathology, Free University Medical Center, de Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands
Tumour cell insensitivity to anticancer drugs frequently appears as multidrug resistance (MDR), associated with overexpression of one or more of a set of at least 10 different molecules, causing reduced drug levels at the intracellular target sites. They include transmembrane transporter proteins such as P glycoprotein, MRP1–9 and BCRP. In addition, the lung-resistance protein, recently identified as the major vault protein, has been associated with MDR. We have generated monoclonal antibodies that specifically recognize most of these proteins, which we are using to try to identify their roles in clinical drug resistance, and also to explore their occurrence in normal human tissues and physiology. Both types of studies will also provide further insights into the molecular features of drugs associated with distinct MDR transporters.
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©2002 The Novartis Foundation
Drug resistance in epilepsy: the role of the blood—brain barrier
N. Joan Abbott, Ehsan U. Khan, Christopher M. S. Rollinson, Andreas Reichel, Damir Janigro, Stephen M. Dombrovski, Michael S. Dobbie and David J. Begley
Blood–Brain Barrier Research Group, Centre for Neuroscience Research, King’s College London, London SE1 1UL, UK and
Cerebrovascular Research, Cleveland Clinic Foundation, Cleveland, Ohio OH 44195,
USA
The blood–brain barrier (BBB) is formed by the endothelial cells lining the brain microvessels. Complex tight junctions linking adjacent endothelial cells make brain capillaries around 100 times tighter than peripheral capillaries to small hydrophilic molecules. As a result, drugs required to act in the brain, including anti-epileptic drugs (AEDs), have generally been made lipophilic, able to cross the brain endothelium via the lipid membranes. However, such lipophilic drugs are potential substrates for efflux carriers of the BBB, particularly P glycoprotein (Pgp), predominantly located on the endothelial luminal membrane. It is estimated that up to 50% of drug candidates may be substrates for Pgp. The barrier phenotype of the brain endothelium is induced and maintained by chemical factors released by brain cells, particularly perivascular astrocytic end feet. In several neuropathological conditions, the BBB is disturbed, either as a result of pathology of the endothelium, or of the cells responsible for barrier induction and maintenance. During epileptic attacks, there may be transient BBB opening in the epileptogenic focus. There is evidence that under such pathological conditions, ‘second line defence’ mechanisms in perivascular glia may be up-regulated, including expression of Pgp and other drug efflux transporters. This complicates interpretation of drug resistance in epilepsy, and therapeutic strategies.
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©2002 The Novartis Foundation
P glycoprotein and the mechanism of multidrug resistance
András Váradi, Gergely Szakács, Éva Bakos and Balázs Sarkadi
Institute of Enzymology, Hungarian Academy of Sciences, Karolina ut 29, Budapest H-1113, and National Institute of Hematology and Immunology, Membrane Research Group of SEB-Hungarian Academy of Science, Daróczi ut 24, Budapest H-1113, Hungary
The human P glycoprotein (MDR1) is an ATP-driven transporter for hydrophobic drugs and causes multidrug resistance in cancer. Our knowledge related to the mechanistic details of the ATP hydrolytic cycle of MDR1 has recently significantly progressed due to studies on the formation of a catalytic intermediate (occluded nucleotide state). According to the most accepted current model, both catalytic sites in MDR1 are active and ATP is hydrolysed alternatively within the two sites. ATP hydrolysis at one site triggers conformational changes within the protein resulting in drug transport, while hydrolysis of a second ATP molecule (at the other site) is required for resetting the initial (‘high-affinity binding’) conformation. The two active sites act in a cooperative manner and experiments support a model where the two ATP binding cassette (ABC) domains form a coupled catalytic machinery. Although no high resolution structure is available as yet, some relevant structural information can be deduced from crystal structures obtained for several bacterial ABC units, and the recently solved bacterial ABC–ABC dimer crystal structures may provide the basis for a better understanding of the intramolecular cross-talk between the two catalytic sites. As intramolecular interactions between various domains of Pgp/MDR1 are essential in regulating both the ATPase and transport activity, compounds perturbing these interactions may interfere with the function of the transporter. Such compounds, as well as various substrate analogues may be useful in modulating multidrug resistance in cancer.
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©2002 The Novartis Foundation
Drug resistance caused by multidrug-resistance associated proteins
Jan Wijnholds
Department of Ophthalmogenetics, The Netherlands Ophthalmic Research Institute, Meibergdreef 47, 1105 BA, Amsterdam and Division of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands
Three types of drug efflux pumps, the multidrug resistance gene 1 (MDR1 or ABCB1)-encoded P glycoprotein, the multidrug resistance-associated protein (MRP or ABCC1) and breast cancer resistance protein (BCRP or ABCG2) may play an important part in the intrinsic or acquired defence of cells against drugs. Recent studies have begun to show the broad tissue distribution and drug substrate specificity of the seven MRP family members (MRP1–7; or ABCC1–6 and ABCC10). MRPs are (multispecific) organic anion transporters, which can transport negatively charged anionic drugs and neutral drugs conjugated to glutathione, glucuronate or sulfate. MRP4 and MRP5 broaden the spectrum of drug resistance to nucleotide analogue drugs. Some MRPs can also transport neutral drugs if co-transported with glutathione. MRP1 and MRP5 are abundant in almost every organ and are prominently present in the brain. High levels of MRP1 are present in the epithelium of the choroid plexus. Using mutant mice lacking a functional Mrp1 gene, we have previously shown the contribution of MRP1 to the blood–CSF (cerebrospinal fluid) drug permeability barrier. Recent studies indicate that the very low levels of MRP1 or MDR1 present in fibroblasts affect their sensitivity to a wide range of clinically important cytotoxic drugs. Even low concentrations of drug transporters may therefore protect cells against
drugs.
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©2002 The Novartis Foundation
Drug resistance in cancer therapy
Susan E. Bates, Clara Chen, Robert Robey, Min Kang, William D. Figg and Tito Fojo
Molecular Therapeutics Section, Medicine Branch, National Cancer Institute, Bethesda, MD 20892, USA
Modulation of P glycoprotein (Pgp) in clinical oncology has had limited success. Contributing factors have included the limitation in our understanding of the tumours in which Pgp overexpression is mechanistically important in clinical drug resistance; the failure to prove that concentrations of modulator achieved in patients were sufficient to inhibit Pgp; and the inability to conclusively prove that Pgp modulation was occurring in tumours in patients. New approaches are needed to determine the clinical settings in which Pgp overexpression plays a major role in resistance. Clinical trials with third generation modulators are ongoing, including trials with the compounds LY335979, R101933 and XR9576. Using the Pgp substrate Tc-99m Sestamibi as an imaging agent, we have seen increased uptake in normal liver and kidney after administration of PSC 833, VX710 and XR9576. These studies confirm that the concentrations of modulator achieved in patients are able to increase uptake of a Pgp substrate. Further, CD56+ cells obtained from patients treated with PSC833 demonstrate enhanced rhodamine retention in an ex vivo assay after administration of the antagonist. Finally, a subset of patients treated with Pgp antagonists show enhanced Sestamibi retention in imaged tumours. These results suggest that Pgp modulators can increase drug accumulation in Pgp-expressing tumours and normal tissues in patients. Using third generation Pgp antagonists and properly designed clinical trials, it should be possible to determine the contribution of modulators to the reversal of clinical drug resistance.
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©2002 The Novartis Foundation
Clinical development of P glycoprotein modulators in oncology
Amit M. Oza
Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9
The last two decades have witnessed dramatic advances into the mechanisms of drug resistance in cancer. The identification of P glycoprotein (Pgp) as a specific mechanism led to the initial hope and expectation that it would be possible to modulate this and increase sensitivity to drug therapy. Clinical trials using first- and second-generation Pgp modulators did establish proof of principle that in some settings, clinical drug resistance could be overcome with the addition of a Pgp modulator—for example, clinical resistance to paclitaxel, a Pgp substrate, in women with ovarian cancer was shown to be overcome in approximately 20% with the addition of PSC 833, a highly effective Pgp modulator. However, evolutionary and adaptive redundancy in resistance mechanisms have tempered clinical results, even with very effective second- and third-generation modulators. The lessons from oncology establish sound methodology for the evaluation of Pgp modulators for safety, tolerability and efficacy in Phase I, II and III clinical trials. This review will focus on some of the early-phase clinical trials with earlier and newer Pgp modulators, either as single agents or in combination with
chemotherapy.
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©2002 The Novartis Foundation
Gene expression profiling of epothilone A-resistant cells
Peter Atadja, Yan Yan-Neale, Harry Towbin, Frank Buxton and Dalia Cohen
Functional Genomics, Novartis Corporation, Summit, NJ 07901, USA
In the current study, we isolated sublines of the human breast adenocarcinoma cell line MDA 435 that exhibited increasing resistance to epothilone A, a microtubule-stabilizing cytotoxic agent. The resistant cells did not express P glycoprotein or multidrug resistance-associated protein (MRP) which are known mediators of multidrug resistance (MDR). Two groups of epothilone A-resistant cells were selected: cells which exhibited low resistance to both epothilone A and taxol, and cells which exhibit low resistance to taxol but high resistance to epothilone A. cDNA microarrays of epothilone A-resistant and taxol-resistant cells were utilized to further characterize epothilone A resistance. Hierarchical clustering of genes according to their levels of expression indicated that majority of genes which were highly expressed in epothilone A-resistant cells but not in taxol-resistant MDR cells encode known interferon-inducible proteins. Genes whose expression increased with increasing epothilone A resistance include microtubule-associated GTPases, cytoskeletal proteins, cell signalling proteins and a drug metabolising enzyme. The majority of the genes that were repressed in both epothilone A- and taxol-resistant cells encode proteins regulating cellular growth signalling mechanisms.
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©2002 The Novartis Foundation
Imaging of P glycoprotein function in vivo with PET
N. H. Hendrikse and W. Vaalburg
PET Center, University Hospital of Groningen, PO Box 30001, 9700 RB Groningen, The Netherlands
P glycoprotein (Pgp) is expressed on cell membranes of various organs in the body, such as the capillary endothelial cells of the brain. Furthermore, Pgp can also be expressed on the cell membrane of tumour cells. Because of Pgp-mediated efflux, tissue levels of several Pgp substrates are lower than in Pgp-negative tissues. Drug levels in Pgp-expressing organs may be increased by modulation of this Pgp-facilitated transport with several compounds, such as cyclosporin A. Up to now, the presence of drug efflux pumps in tissues could only be examined at the mRNA and protein level. However, this gives no insight as to the important question of the functionality of these drug efflux pumps. Information about the transport function of Pgp and the effect of modulating this function may improve the therapeutic treatment of these patients. Positron emission tomography (PET) gives us a unique opportunity to study non-invasively (patho)physiological dynamic processes in vivo. We have therefore developed and validated a method for studying Pgp-mediated transport and its modulation in vivo with PET.
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©2002 The Novartis Foundation
Animal models of drug-resistant epilepsy
Wolfgang Löscher
Department of Pharmacology, Toxicology, and Pharmacy, School of Veterinary Medicine, Bünteweg 17, 30559 Hannover, Germany
It is not known why and how epilepsy becomes drug resistant in 20–30% of patients, while other patients with seemingly identical seizure types can achieve control of seizures with medication. An animal model of epilepsy allowing selection of pharmacoresistant and pharmacosensitive subgroups of animals would be a valuable tool to study mechanisms of pharmacoresistance and to develop more effective treatment strategies. Only a few models are available which mimic patterns of drug resistance in humans with epilepsy. One model seems to be particularly interesting: amygdala-kindled rats. In this model in Wistar rats, animals which do not respond to repeated or chronic administration of antiepileptic drugs (non-responders) can be separated from animals in whom antiepileptics are effective (responders). Hence, pharmacoresistant subgroups of kindled rats provide a unique tool to study why seizures become intractable, particularly because pathophysiological processes in these resistant rats can be directly compared with those of kindled rats that respond to treatment. By using this model, we have recently shown that both the individual genetic background and kindling-induced processes determine whether a rat becomes a responder or non-responder to anticonvulsant treatment after kindling. We are currently studying the cellular mechanisms underlying the development of drug-resistant kindled seizures.
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©2002 The Novartis Foundation
Drug resistance in epilepsy: human epilepsy
S. M. Sisodiya, W.-R. Lin, B. N. Harding, M. V. Squier and M. Thom
Epilepsy Research Group, University Department of Clinical Neurology, Department of Neuropathology, Institute of Neurology, and Department of Neuropathology, Institute of Child Health, University College London, Queen Square, London, and
Department of Neuropathology, Radcliffe Infirmary, Oxford, UK
The basis of drug resistance in human epilepsy is not understood. Parallels with resistance in cancer suggest that drug resistance proteins may have a role. To examine this possibility, we have studied human brain tissue containing pathologies capable of causing refractory epilepsy. Using immunohistochemistry for P glycoprotein (Pgp) and multidrug-resistance associated protein 1 (MRP1), we examined both pathological tissue and control tissue. We demonstrate expression of Pgp and MRP1 in glia from cases of malformation of cortical development studied both before and after the onset of epilepsy, as well as in cases of hippocampal sclerosis and dysembryoplastic neuroepithelial tumours. In one particular type of malformation, we also demonstrate that dysplastic neurons express MRP1. The pattern of immunolabelling suggests overexpression is concentrated particularly around vessels in most of the pathologies. The timing shows that expression may be constitutive in some pathologies. These findings suggest that drug resistance proteins may contribute to drug resistance in refractory epilepsy.
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©2002 The Novartis Foundation
Cellular mechanisms of pharmacoresistance in slices from epilepsy surgery
R. A. Diesz
Department of Cell and Neurobiology, Institute of Anatomy, Charité, 10098 Berlin
Slices of human cortical tissue from epilepsy surgery were investigated with intracellular recordings to elucidate the mechanisms contributing to augmented synaptic excitation and to repetitive activity. The analysis of single synaptic potentials revealed, amongst other differences to rodent cortex, a disturbance of GABAA inhibition, namely depolarizing responses. A tentative ionic mechanism, impaired KCl outward-transport (KCC2), was evaluated in a rat model (0-Mg hyperexcitability). The observed down-regulation of KCC2 mRNA after 0-Mg-ACSF exposure of slices may contribute to the depolarizations by GABA. The factors enabling repetitive activity were addressed with a paired-pulse paradigm. In slices from epilepsy surgery, synaptic responses were virtually constant with interstimulus intervals between 100 and 1000ms. Tiagabine markedly prolonged the effects of released GABA at GABAA receptors, but paired-pulse behaviour was only slightly affected. We demonstrate that bicuculline-induced paroxysmal activity of rat cortex is frequency-limited (to about <1Hz) by presynaptic GABAB receptors. The lack of frequency limitation of synaptic events suggests an impaired GABA receptor function in the human epileptogenic cortex. The data are discussed towards the pivotal role of KCl transport in epileptic disorders of various origins and the role of GABAB receptors in the frequency limitation of paroxysmal activity.
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©2002 The Novartis Foundation