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Expression and function of ABC multidrug transport proteins in patients with non-responsive focal epilepsy

  • About 30 % of epileptic patients are non-responsive to multidrug antiepileptic therapy. One of non-responsiveness in epilepsy hypothesis claims that non-responsiveness occurs because of reduced access of antiepileptic drugs to their targets, as a result of increased efflux of antiepileptic drugs away from these targets. Transporters believed to be involved in non-responsiveness in epilepsy are mainly but not exclusively the members of the ABC superfamily including P-gp (MDR1, ABCB1), MRP1 (ABCC1), MRP2 (ABCC2) and others. These proteins are normally found in the blood-brain barrier and the blood-cerebrospinal fluid barrier where they function as protectors. There is emerging evidence that P-gp, MRP1 and MRP2 are up-regulated in epileptogenic brain tissue. The risk of non-responsiveness could be related also to the MDR1 or MRP2 gene polymorphisms. We hypothesised that changes in expression and function of multidrug transporters involved in non-responsiveness of epilepsy might be detectable not only in the brain but also in other tissues such as lymphocytes. Therefore we evaluated the expression of MDR1, MRP1 and MRP2 and function of P-gp in lymphocytes in patients with epilepsy and healthy subjects. Three groups of epileptic patients and 15 healthy subjects as a control group were included in the study. The patients’ group was defined as follows: Monotherapy – patients treated with carbamazepine monotherapy, without seizures - corresponded to group responders. Combined therapy – patients after monotherapy (two different medicines have been tried) and combined therapy (two trials of combined therapy), not free of seizures. Monotherapy and combined therapy groups each embraced 15 patients. Neurosurgery – patients who had undergone neurosurgery, afterwards were or were not additionally treated with carbamazepine, with or without seizures. This group comprised 24 patients. Combined therapy and neurosurgery groups composed the group of non-responders. The mRNA expression of MRP1, MRP2 and MDR1 by means of quantitative real-time PCR as well as MRP2 and P-gp protein content by Western blot in lymphocytes was measured. For P-gp functional analysis rhodamine efflux from lymphocytes and natural killer (NK) cells was performed. The influence of the polymorphisms C3435T, G2677T/A in the MDR1 gene and C24T, G1249A, C3972T in the MRP2 gene for the transporters expression, function and their association with non-responsive epilepsy phenotype was investigated. Our results showed that MRP1 expression in lymphocytes was significantly lower in epileptics than in healthy subjects. Non-responders had lower MRP1 mRNA content in lymphocytes than responders. We did not find any difference in MRP2 expression between epileptics and healthy volunteers. MRP2 mRNA levels in lymphocytes were higher in non-responders than in responders. However, at protein level epileptic patients had significantly lower MRP2 content in lymphocytes than controls. MRP2 protein content did not differ in responders and non-responders. There was no reliable correlation between MRP2 mRNA expression and MRP2 protein content in lymphocytes. Epileptics had significantly lower MDR1 expression in lymphocytes than healthy individuals. MDR1 expression was decreasing according to the consumption of antiepileptic drugs and seizures frequency: patients after neurosurgery had significantly lower MDR1 expression than patients after combined therapy and monotherapy. MDR1 expression was significantly lower in non-responders than in responders. At protein level epileptics had lower P-gp content than controls. Detected P-gp amount in lymphocytes did not differ between responders and non-responders. Rhodamine efflux from lymphocytes and NK cells did not differ significantly between epileptics and healthy subjects, but it was higher in patients after neurosurgery than in patients after monotherapy. Rhodamine efflux from NK cells, which are known to express the highest levels of P-gp, was significantly higher in non-responders than in responders. In this study, we showed that MRP1 mRNA expression in lymphocytes was significantly correlated to its expression in the brain. We detected also a significant co-correlation between MRP1 expression in the hippocampus and MDR1 expression in lymphocytes. We found no evidence regarding the impact of the MDR1 polymorphisms on mRNA expression, P-gp content and rhodamine efflux from lymphocytes. Our data showed lack of evidence regarding the impact of the MRP2 polymorphisms on mRNA expression and protein content. We did not detect any association between MDR1 or MRP2 polymorphisms and non-responsiveness in epilepsy or epilepsy in the main. In conclusion, our results suggest that lymphocytes are an appropriate surrogate for studies on changes of multidrug transporters expression in epilepsy. Lymphocytes as an easily accessible tissue might serve as a marker for responsiveness to antiepileptic drug therapy in epilepsy studies.
  • In dieser klinischen Studie untersuchten wir die Expression und Funktion von ABC-Transportproteinen in Lymphozyten bei Patienten mit fokaler Epilepsie und unterschiedlichen Therapieerfolg im Vergleich zu gesunden Probanden. Der Gehalt an MRP1 und MDR1 in Lyphozyten therapieresistenter Patienten war signifikant vermindert gegenüber nichtresistenenten Patienten und gesunden Kontrollprobanden. Bei MRP 2 zeigte sich ein signifikanter Unterschied zwischen Patienten mit Epilepsie und Gesunden. Die Proteinfunktion von P-gp korreliert nicht mit der Expression von MDR1. Es zeigte sich eine signifikante Korrelation von MRP1 im Gehirn (Gyrus temporalis) und Lymphozyten, weiter auch eine Co-Korrelation von MRP1 im Gehirn (Hippocampus) und MDR1 in Lymphozyten bei Patienten mit therapiersistenter Epilepsie. Unsere Arbeit lässt vermuten, daß Lymphozyten ein geeignetes, gut verfügbares Substrat für Transporteruntersuchung bei Epilepsiestudien und ein Marker für das Ansprechen einer Therapie mit antiepileptischen Medikamenten sein könnten.

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Author: Snieguole Upermonaite
Title Additional (German):Expression und Funktion von ABC-Transportern bei Patienten mit therapieresistenter fokaler Epilepsie
Advisor:Prof. Dr. Werner Siegmund
Document Type:Doctoral Thesis
Date of Publication (online):2006/11/14
Granting Institution:Ernst-Moritz-Arndt-Universität, Medizinische Fakultät (bis 2010)
Date of final exam:2006/10/19
Release Date:2006/11/14
Tag:ABC-transporters; clinical pharmacology; clinical study; epilepsy; lymphocyte
GND Keyword:Klinische Pharmakologie, Epilepsie, ABC-Transporter, Lymphozyt, Rhodaminfarbstoff, Immunoblot, Real time quantitative PCR, Klinisches Experimen
Faculties:Universitätsmedizin / Institut für Pharmakologie
DDC class:600 Technik, Medizin, angewandte Wissenschaften / 610 Medizin und Gesundheit