Modulating-P-glycoprotein-regulation-Future-perspectives-for-pharmacoresistant-epilepsies_2010_Epile
Modulating P-glycoprotein regulation:Future perspectives for
pharmacoresistant epilepsies?
Heidrun Potschka
Institute of Pharmacology,Toxicology,and Pharmacy,Ludwig-Maximilians-University,Munich,Germany
S UMMARY
Enhanced brain ef?ux of antiepileptic drugs by the blood–brain barrier transporter P-glycoprotein is discussed as one mechanism contributing to pharmacoresistance of epilepsies.P-glycoprotein overexpression has been proven to occur as a consequence of seizure activity.Therefore,blocking respective signaling events should help to improve brain penetration and ef?cacy of P-glyco-protein substrates.A series of recent studies revealed key signaling factors involved in seizure-associated transcrip-tional activation of P-glycoprotein.These data suggested several interesting targets,including the N -methyl-D -aspartate (NMDA)receptor,the in?ammatory enzyme cyclooxygenase-2,and the prostaglandin E2EP1receptor.These targets have been further evaluated in rodent mod-els,demonstrating that targeting these factors can control P-glycoprotein expression,improve antiepileptic
drug brain penetration,and help to overcome pharmaco-resistance.In general,the approach offers particular advantages over transporter inhibition as it preserves basal transporter function.In this review the different strategies for blocking P-glycoprotein upregulation,including their therapeutic promise and drawbacks are discussed.Moreover,pros and cons of the approach are compared to those of alternative strategies to overcome transporter-associated resistance.Regarding future perspectives of the novel approach,there is an obvious need to more clearly de?ne the clinical relevance of transporter overexpression.In this context current efforts are discussed,including the development of imaging tools that allow an evaluation of P-glycoprotein function in individual patients.
KEY WORDS:Pharmacoresistance,Blood–brain barrier,Multidrug transporter,Cyclooxygenase-2,Epilepsy,NMDA receptor.
Elucidation of the mechanisms of pharmacoresistance casts hope that the gain in knowledge renders a basis for novel therapeutic concepts.Three major hypotheses are dis-cussed to explain therapeutic failure,which is likely to con-stitute a multifactorial problem.The intrinsic severity hypothesis suggests that neurobiologic factors that confer increased disease severity lead to drug refractoriness (Rog-awski &Johnson,2008).According to the target hypothesis,specific alterations in target sites reduce affinity or efficacy of antiepileptic drugs at their targets (Remy &Beck,2006).In contrast,the transporter hypothesis relates to pharmaco-kinetic aspects,which suggest that overexpressed efflux transporters at the blood–brain barrier limit brain penetration of antiepileptic drugs (Sisodiya,2003;Loscher &Potschka,2005).The latter hypothesis was based initially on the obser-vation that different multidrug transporters are expressed at high levels in brain capillary endothelial cells in tissue
dissected from patients with pharmacoresistant epilepsy (Tishler et al.,1995;Dombrowski et al.,2001;Sisodiya et al.,2002;Aronica et al.,2004;Lazarowski et al.,2004;Kubota et al.,2006;Ak et al.,2007).A series of investiga-tions in rodent models revealed that seizure activity upregulates blood–brain barrier efflux transporters including P-glycoprotein (=ABCB1),members of the ABCC trans-porter family,and breast cancer resistance protein (=ABCG2)(Rizzi et al.,2002;Seegers et al.,2002a,b;van Vliet et al.,2004,2005;Volk et al.,2005;Hoffmann et al.,2006;Liu et al.,2007).In vitro and in vivo transport assays have indicated that several antiepileptic drugs are substrates of P-glycoprotein,and that some antiepileptic drugs are transported by ABCC transporters (Loscher &Potschka,2005).However,controversial data have been reported for several antiepileptic drugs (Anderson &Shen,2007),so more research is obviously needed to determine which an-tiepileptic drugs are affected by overexpressed transporters.(For a discussion of the data on transport of antiepileptic drugs by human P-glycoprotein see Future Perspectives.)Moreover,putative species differences in the substrate spec-trum of the transporters need to be taken into account when extrapolating from rodent data to the clinical situation.A functional role in pharmacoresistance has been experimen-
Accepted March 5,2010;Early View publication May 14,2010.
Address correspondence to Heidrun Potschka,Institute of Pharmacol-ogy,Toxicology,and Pharmacy,Ludwig-Maximilians-University,Koeniginstr.16,D-80539Munich,Germany.E-mail:potschka@pharmtox.vetmed.uni-muenchen.de
Wiley Periodicals,Inc.
a2010International League Against Epilepsy Epilepsia ,51(8):1333–1347,2010
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CRITICAL REVIEW AND INVITED COMMENTARY
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tally demonstrated for P-glycoprotein in a series of studies showing that upregulation of P-glycoprotein is associated with reduced brain penetration of antiepileptic drugs(Rizzi et al.,2002;Marchi et al.,2006;Hocht et al.,2007;Liu et al.,2007;van Vliet et al.,2007b;Hocht et al.,2009;)and that a correlation exists between P-glycoprotein expression rates and pharmacosensitivity(Potschka et al.,2004;Volk &Loscher,2005).Moreover,targeting P-glycoprotein by modulators can enhance the efficacy of antiepileptic drugs. In particular,coadministration of the first-generation inhibi-tor verapamil proved to potentiate the anticonvulsant effi-cacy of the antiepileptic drug oxcarbazepine(Clinckers et al.,2005).The third-generation modulator tariquidar increased the efficacy of phenytoin in a chronic rat epilepsy model and helped to overcome pharmacoresistance to phe-nobarbital in chronic rat epilepsy models(Brandt et al., 2006;van Vliet et al.,2006).When drawing conclusions from these data,consideration must be given to the possibil-ity that a limited specificity of the modulators might bias the impact of the add-on treatment on antiepileptic drug efficacy. Whereas first-and second-generation inhibitors can exert additional pharmacodynamic and pharmacokinetic effects, third-generation inhibitors are considered fairly specific.How-ever,for the third-generation inhibitor tariquidar evidence exists that it can also affect the efflux transporter ABCG2. Inhibition of P-glycoprotein transport function is an obvious strategy that might be further developed in transla-tional approaches.However,in this context it needs to be emphasized that respective evidence with a third-generation inhibitor has so far only been reported for phenobarbital and phenytoin.Moreover,long-term inhibition of this trans-porter needs to take into account that P-glycoprotein serves as a protective mechanism and gatekeeper in several blood-tissue barriers as well as hematopoietic cells(Huls et al., 2009).In addition to limiting access of harmful xenobiotics to sensitive tissues or cells,P-glycoprotein also accelerates extrusion of xenobiotics based on its efflux function in the liver and kidneys.Therefore,alternate approaches that leave basal transporter expression and function unaffected might offer advantages for tolerability and safety issues.Prevent-ing seizure-associated transporter upregulation might offer an intriguing alternate approach to overcoming transporter-associated pharmacoresistance.Development of approaches that target transporter regulatory pathways of course requires an in-depth understanding of the signaling mecha-nisms that contribute to activation of transporter expression in the epileptic brain.
Identi?cation of Targets
Involved in P-Glycoprotein
Regulation
As already stated,experimental data indicate that seizure activity is the main factor upregulating P-glycoprotein in the epileptic brain(Rizzi et al.,2002;Seegers et al., 2002a,b;van Vliet et al.,2004,2005;Volk et al.,2005; Hoffmann et al.,2006;Liu et al.,2007).Some studies have indicated that antiepileptic drugs might contribute to the induction.In the coriaria lactone-kindling model,treatment with carbamazepine or valproic acid was associated with higher blood–brain barrier P-glycoprotein expression rates as compared to treatment with lamotrigine or topiramate (Wang-Tilz et al.,2006).However,the differences observed in this study might be related to varying effects on seizure control.Lombardo et al.(2008)reported that carba-mazepine,phenobarbital,and phenytoin induce P-glycopro-tein and other transporters in rat brain endothelial cell lines via an interaction with the pregnane X receptor and the constitutive androstane receptor.In apparent contrast, Ambroziak et al.(2010)did not observe effects of these antiepileptic drugs on expression and functionality of P-gly-coprotein in the rat brain endothelial cell line GPNT. Therefore,whereas seizure-associated induction of P-glyco-protein has been demonstrated in a reproducible manner, definite conclusions regarding the impact of antiepileptic drugs require further research efforts.
The elucidation of signaling events that mediate seizure-associated P-glycoprotein upregulation has been initiated based on the selection of initial candidate factors.Glutamate was an obvious candidate factor as it reaches excessive concentrations in the extracellular space during epileptic seizures(Holmes,2002).Moreover,Zhu and Liu(2004) reported that glutamate can affect P-glycoprotein https://www.360docs.net/doc/404194636.html,ing isolated brain capillaries,we confirmed the specific role of this excitatory neurotransmitter in the regu-lation of P-glycoprotein.Exposing isolated rat and mouse brain capillaries to glutamate increased the expression and transport function of P-glycoprotein in a concentration-dependent manner(Bauer et al.,2008).Blockade of tran-scription or translation counteracted this effect of glutamate, suggesting that the impact of glutamate on P-glycoprotein depends on de novo protein synthesis.The excitatory neuro-transmitter glutamate is known to signal via different iono-tropic and metabotropic receptors in neurons(Meldrum& Rogawski,2007).N-Methyl-D-aspartate receptors(NMDA-Rs)represent one subtype of ionotropic glutamate receptor that is specifically activated by NMDA(Meldrum&Rog-awski,2007).In response to glutamate binding,NMDA-Rs mediate an influx of Na+and Ca2+into the cells and of K+ out of the cells(Sattler&Tymianski,2000).Earlier studies have already demonstrated a function of NMDA-R in brain capillary endothelial cells(Koenig et al.,1992;Zhu&Liu, 2004).Incubation of rat brain capillaries with NMDA increased P-glycoprotein expression and transport function (Bauer et al.,2008).Moreover,the competitive NMDA-R blocker MK-801prevented the effect of glutamate on P-gly-coprotein in the same experimental set-up.These data identified the NMDA-R as an endothelial key factor driving P-glycoprotein expression.
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The course of events in signaling cascades might gener-ally be affected by the cellular environment.Therefore,we aimed to substantiate the role of glutamate under more complex in vivo conditions(Bauer et al.,2008).In line with its function as an initial signaling factor,hippocampal microinjections of glutamate at concentrations that did not exert behavioral or electrographic seizure activity resulted in a significant increase in P-glycoprotein expression(Bauer et al.,2008).
The confirmation of glutamate and the NMDA-R as sig-naling factors in P-glycoprotein regulation initiated further efforts to identify downstream events.The rise in intracellu-lar calcium mediated by NMDA-R activation is known to activate several enzymatic pathways in neurons including phospholipase A2(Mishra&Delivoria-Papadopoulos, 1999).Phospholipase A2releases arachidonic acid from the cell membrane,which is then processed by cyclooxygen-ase-2(COX-2)to generate prostanoids.Our recent investi-gations revealed that the same signaling pathway is activated in brain capillary endothelial cells involving COX-2as a central downstream factor(Bauer et al.,2008). Both the nonselective COX inhibitor indomethacin as well as the selective COX-2inhibitor celecoxib counteracted P-glycoprotein induction by glutamate exposure of isolated rat brain capillaries.The central signaling function of COX-2was undoubtedly confirmed by experiments in mouse capillaries isolated from COX-2deficient animals. As discussed below a series of in vivo experiments further substantiated the key signaling function of COX-2. Regarding the identification of further targets in the sig-naling pathway,it was considered that prostaglandin E2 (PGE2)constitutes the major product of COX-2signaling in the brain(Zhang&Rivest,2001).PGE2signals via differ-ent G-protein–coupled metabotropic receptors including EP1,EP2,EP3,and EP4(Hata&Breyer,2004).Each of these receptors is associated with different signal transduc-tion mechanisms(Hata&Breyer,2004).Among these receptors,the EP1receptor is the only one that can be considered as a putative target in the context of transporter-associated pharmacoresistance.EP2,EP3,and EP4antago-nists can aggravate neurodegeneration(Bilak et al.,2004; McCullough et al.,2004;Ahmad et al.,2005),so that blockage of these receptors would be associated with unac-ceptable consequences in the epileptic brain.In apparent contrast,blocking EP1receptors proved to exert neuropro-tective effects in a rodent model of brain ischemia (Suganami et al.,2003;Kawano et al.,2006).Therefore,we focused our experimental efforts on whether EP1receptors contribute to the signaling events that regulate P-glycopro-tein in response to glutamate.Substantiating its functional role as a downstream signaling factor,antagonism of the EP1receptor abolished the effect of glutamate on brain capillary P-glycoprotein expression(Pekcec et al.,2009). In summary,the investigations revealed a glutamate/ NMDA-R/COX-2/EP1-R signaling cascade that results in transcriptional activation of P-glycoprotein associated with epileptic seizure activity(Fig.1).Further research efforts currently focus on events that are involved in EP1-R down-stream signaling.
Targeting P-Glycoprotein
Regulation in the Epileptic Brain:Ef?cacy and Tolerability The elucidation of signaling key events that drive P-gly-coprotein expression in response to seizure activity sug-gested several targets including the NMDA-R,COX-2,and the EP1-R.
Regarding its future promise,further validation of these targets involves the following requirements;targeting of the regulatory factor:(1)prevents seizure-associated upregula-tion of P-glycoprotein expression;(2)normalizes P-glyco-protein expression in individuals with recurrent seizures;
(3)improves brain penetration of antiepileptic drugs;
(4)improves efficacy of antiepileptic drugs;(5)helps to overcome pharmacoresistance;and(6)must be tolerable in patients with epilepsy.The current status of the efficacy testing for the different targets is summarized in Table1. Moreover,tolerability issues that should be considered are listed.
Experimental data suggest the NMDA-R as a target for interference with P-glycoprotein upregulation(Bauer et al., 2008).MK-801is a noncompetitive NMDA-R antagonist that binds at a site within the receptor ion channel (Woodruff et al.,1987).Coincubation of rat capillaries with MK-801efficaciously prevented P-glycoprotein induction by glutamate(Bauer et al.,2008).Supporting this observa-tion,pretreatment of rats with MK-801counteracted P-glycoprotein upregulation in a status epilepticus model (Bankstahl et al.,2008a).The NMDA-R complex has been discussed intensely as a target for epilepsy treatment(Kohl &Dannhardt,2001),promising neuroprotective effects.In support of its neuroprotective effect,the noncompetitive NMDA-R antagonist MK-801efficaciously prevented neuronal cell loss in the hippocampus and piriform cortex in rat kainic acid model(Brandt et al.,2003).Moreover,anti-convulsant effects have been reported for competitive and noncompetitive antagonists of NMDA-R.All these data would rather favor the use of NMDA-R antagonists as an add-on treatment with antiepileptic drugs aiming to optimize brain penetration and efficacy of the antiepileptic drug.However,when considering the NMDA-R as a target, tolerability issues also need to be considered.Noncompeti-tive NMDA-R antagonists such as phencyclidine(PCP)or MK-801induce a behavioral PCP-like syndrome in rodents consisting of hyperlocomotion,stereotypies,and pronounced motor impairment(Tricklebank et al.,1989). Competitive antagonists were considered advantageous,as comparable effects are observed only at doses higher than
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those producing therapeutically relevant effects in naive rodents (Loscher &Honack,1991;Wlaz et al.,1998).How-ever,it has been reported that a competitive NMDA-R antagonist induces the behavioral syndrome when adminis-tered in a chronic rat epilepsy model at low to medium doses (Loscher &Honack,1991;Wlaz et al.,1998).Therefore,epileptogenesis seems to render the brain more susceptible to PCP-like behavioral effects.These PCP-like behavioral effects are considered to reflect psychotomimetic and rein-forcing effects in humans.In line with the experimental data,clinical testing of a competitive NMDA-R antagonist was associated with severe adverse effects including confu-sion,disorientation,ataxia,and sedation in epilepsy patients,whereas the compound was well tolerated in healthy volunteers (Sveinbjornsdottir et al.,1993).There-fore,based on these preclinical and clinical data,NMDA-R antagonists that bind in the ion channel or competitively inhibit glutamate binding cannot be considered suitable compounds for add-on treatment in epilepsy patients.
Taking these aspects into consideration,research has more intensely focused on alternate targets in the P-glyco-protein regulatory cascade such as COX-2.With two major
isoforms,COX enzymes catalyze the first rate-limiting step in prostanoid synthesis (Ueno et al.,2005).A large selec-tion of nonsteroidal antiinflammatory drugs is clinically available,which efficaciously inhibits COX enzymes (Patrono &Baigent,2009).Thereby compounds can differ significantly in their selectivity for one or the other COX isoform.Substantiating our approach to target NMDA-R downstream events,the nonselective COX-inhibitor indo-methacin prevented the seizure-associated increase in capil-lary P-glycoprotein expression in the pilocarpine status epilepticus model in rats (Bauer et al.,2008).Based on in vitro data that pointed to COX-2as the responsible isoform,we tested the hypothesis of whether comparable effects are obtained with a selective COX-2inhibitor.In support of this hypothesis,the selective COX-2inhibitors celecoxib (Fig.2),NS-398,and SC-58236prevented the seizure-mediated transcriptional activation of P-glycoprotein in rat status epilepticus models (Zibell et al.,2009;van Vliet et al.,2010).The effect of all compounds was convincing,with P-glycoprotein expression kept at control levels in dif-ferent brain regions.Thereby,it needs to be considered that a status epilepticus induced by the cholinomimetic
pilocar-
Figure 1.
Extracellular concentrations of glutamate increase during epileptic seizures.Glutamate can signal via endothelial NMDA receptors to activate an intracellular cascade that upregulates P-glycoprotein (Bauer et al.,2008).Ca 2+in?ux via the NMDA receptor is known to activate phospholipase A2,which can release arachidonic acid from the cell membrane.Therefore,Ca 2+might represent the link that drives activation of arachidonic acid signaling.The in?ammatory enzyme cyclooxygenase-2was clearly substantiated as one key down-stream effector that processes arachidonic acid (Bauer et al.,2008;Zibell et al.,2009).Prostaglandin E2as the main end product of cy-clooxygenase-2proved to act via the endothelial EP1receptor (Pekcec et al.,2009).Downstream events of EP1receptors still need to be identi?ed,which then drive transcriptional activation of the P-glycoprotein encoding gene.Epilepsia ILAE
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pine or by electrical stimulation represents a very strong trigger of P-glycoprotein overexpression (Bauer et al.,2008).From a clinical point of view,it is of specific interest whether targeting of COX-2not only prevents P-glycopro-tein upregulation when seizure activity is induced in naive animals,but also controls and downregulates P-glycopro-tein when applied to animals with spontaneous recurrent sei-zures that already exhibit P-glycoprotein overexpression.Testing of the efficacy of COX-2inhibitors was performed in a chronic rodent model,in which P-glycoprotein overex-pression has been reported previously (van Vliet et
al.,
Figure 2.
Representative examples of experiments in the rat pilocarpine status epilepticus model that demonstrate the control of P-gly-coprotein expression based on targeting of its regulation.The COX-2inhibitor celecoxib and the prostaglandin E2EP1recep-tor antagonist SC-51089prevent seizure-induced P-glycopro-tein upregulation (Pekcec et al.,2009;Zibell et al.,2009).The graphs show data from a quantitative analysis of P-glycoprotein immunostaining in the hippocampal hilus and dentate gyrus of vehicle-treated control rats (Vehicle),of vehicle-treated rats with status epilepticus (SE),and of celecoxib-or SC-51089-treated rats with status epilepticus (SE +celecoxib/SC51089).Data are given as mean ±standard error of the mean (SEM).Statistical comparisons:*Signi?cantly higher than controls,p <0.05.Scale bar =50l m.Epilepsia ILAE
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2004,2007b).Subchronic COX-2inhibition controlled P-glycoprotein expression despite recurrent seizure activity (van Vliet et al.,2010).These data substantiate that COX-2inhibition can block repeated induction of P-glycoprotein by ongoing seizure activity,thereby allowing P-glycopro-tein to return to control levels.Enhanced P-glycoprotein expression in chronic epileptic rats was associated with a significant reduction in the brain penetration of the antiepi-leptic drug phenytoin.Importantly,the brain delivery of phenytoin was significantly enhanced by subchronic COX-2inhibition in rats with recurrent seizure activity (van Vliet et al.,2010).These data provided evidence that COX-2inhibition may indeed help to increase concentrations of an-tiepileptic drugs at the target sites in the epileptic brain.The major question was whether the control of P-glycoprotein expression and improvement of antiepileptic drug brain penetration is sufficient to restore antiepileptic drug effi-cacy.This question was addressed using a chronic rodent model with selection of phenobarbital responders and non-responders among rats with spontaneous recurrent seizures.In this experimental approach,pretreatment with the COX-2inhibitor celecoxib efficaciously restored the anticonvul-sant activity of phenobarbital in rats that failed to exhibit a relevant response before celecoxib treatment (Schlichtiger et al.,2010).Whereas P-glycoprotein expression rates in phenobarbital nonresponders selected with this experimen-tal approach significantly exceed P-glycoprotein expression rates in responders (Volk et al.,2006),celecoxib treatment equalized P-glycoprotein expression in these groups (Sch-lichtiger et al.,2010).These data further support COX-2inhibition as a novel therapeutic concept to overcome phar-macoresistance in epilepsies.
Taking completely different therapeutic considerations into account,COX enzymes have already been suggested as targets for adjunctive therapies in epilepsy (Takemiya et al.,2007;Kulkarni &Dhir,2009).COX-2inhibition might have added value in addition to its effects on P-glycoprotein and on antiepileptic drug brain penetration.First,evidence exists that COX-2contributes to excitotoxic brain damage in the epileptic brain,whereas COX-2overexpression accel-erates neuronal apoptosis (Mirjany et al.,2002),inhibition,or genetic deficiency of COX-2resulting in neuroprotection in different rodent epilepsy models (Kunz et al.,2005;Kim &Jang,2006;Takemiya et al.,2006).Second,some studies reported an anticonvulsant effect of COX-2inhibition in dif-ferent rodent seizure and epilepsy models (for review see (Kulkarni &Dhir,2009)).An overview of respective litera-ture comprises about 40publications.The vast majority of these studies describe nonselective COX inhibition,selec-tive COX-2inhibition,or genetic COX-2deficiency as exerting beneficial effects in epilepsy models or without impact on seizure activity and its consequences (for review see (Kulkarni &Dhir,2009)).However,a small number of studies produced evidence that inhibition of COX enzymes can also cause detrimental effects.Akarsu et al.(2006)reported that valeryl salicylate facilitated seizures induced by the chemoconvulsant pentylenetetrazole.In line with these data,an aggravation of seizure activity in the kainic
Table https://www.360docs.net/doc/404194636.html,parison of different approaches to overcome P-glycoprotein-associated pharmacoresistance
Approach
Ef?cacy:experimental evidence
Drawbacks and limitations
References Improvement of AED ef?cacy
Overcoming of pharmacoresistance
Target P-glycoprotein regulation ++Target-speci?c adverse effects (see Table 1)
Pekcec et al.,2009Schlichtiger et al.,2010Modulation of transport function
+
+
Interferes with basal transporter function as a protective mechanism Brandt et al.,2006van Vliet et al.,2006Clinckers et al.,2005RNA interference n.t.n.t.
Interferes with basal transporter function as a protective mechanism;limited downregulation ef?cacy Fuest et al.,2009
Bypassing Intranasal
+n.t.
Lack of convincing evidence that intranasal administration by-passes the blood–brain barrier
Czapp et al.,2008reviewed in Merkus &van den Berg,2007Blood–brain barrier opening n.t.n.t.Not applicable due to epileptogenic effects –
Intracerebral administration +n.t.Invasive approach;restricted distribution Reviewed in Barcia and Gallego,2009
Nanoparticle encapsulation
+
n.t.
Problems with restricted ef?cacy of delivery
Reviewed in Bennewitz &Saltzman,2009
Overview on different strategies to overcome P-glycoprotein-associated pharmacoresistance.The current knowledge regarding ef?cacy of the different approaches in rodent models is summarized,and respective references are listed.For a detailed discussion of intracerebral administration and nanoparticle encap-sulation the reader is referred to recent comprehensive reviews.The most important drawbacks and limitations are given for each approach.AED,antiepileptic drug;n.t.,not tested.
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acid model of mice has been reported with different nonste-roidal antiinflammatory drugs,including the COX-2inhibi-tors celecoxib and NS-398(Baik et al.,1999;Kim et al., 2001,2008).In COX-2knockout mice NMDA-induced sei-zures reached higher severity scores and caused more pro-nounced neuronal cell loss compared to seizures elicited in wild-type mice(Toscano et al.,2008).These reports sug-gest that,depending on the individual conditions,COX-2 inhibition might also exert detrimental effects on seizure control or on the neuropathologic consequences of seizures which,of course,raises concern regarding clinical applica-tions.The reasons for the divergent data still need to be revealed in detail.A more precise future analysis is neces-sary to allow definition of specific individual risk factors that predispose to adverse effects of COX-2inhibition.
In addition to the specific effects in epileptic individuals, general adverse effects of COX-2inhibitors require further consideration.Selective COX-2inhibitors have been designed to develop a novel class of antiinflammatory drugs with an advantageous risk–benefit ratio(Brune,2004).The general drug development concept considered COX-2as an isoform,which plays a major role as an inducible enzyme upregulated by pathophysiologic events(Brune,2004).The developmental concept has not been wholly substantiated, as an increased incidence of cardiovascular and cerebrovas-cular events have been implicated in long-term use of COX-2inhibitors(Brune,2004)and has resulted in with-drawal of some COX-2inhibitors from the market.Selec-tive COX-2inhibitors can decrease the synthesis of vasodilatory,antiaggregatory prostacyclin.However,owing to their lack of action on COX-1,these compounds cannot decrease production of thromboxane A2,which functions in platelet aggregation(Laufer,2004).An increase in the risk for cardiovascular and cerebrovascular events might,there-fore,be related to an imbalance between antithrombotic and prothrombotic eicosanoids caused by selective inhibition of COX-2(Laufer,2004).Regarding gastrointestinal side effects including ulceration,evidence exists that COX-2 inhibitors offer advantages over nonselective COX inhibi-tors(Laufer,2004).However,it needs to be noted that COX-2inhibitors can also cause or promote gastrointestinal damage(Peskar,2005).In particular,COX-2inhibition delays the healing of gastrointestinal ulcers(Peskar,2005). In addition,COX-2might compromise renal function as also indicated by symptoms in COX-2deficient mice(Seta et al.,2009).Decreased urinary sodium and water excretion can result in hypertension or can destabilize blood pressure control in patients treated with antihypertensive agents (Brune,2004).Considering these safety issues,the imple-mentation of COX-2inhibitors as an add-on treatment in patients with pharmacoresistant epilepsy would require sev-eral precautions,including careful patient selection,exclu-sion of patients at risk,and careful monitoring,including repeated evaluation of laboratory parameters.The tolerabil-ity of the approach can be increased by establishing an intermittent add-on treatment.In chronic epileptic rats,a 2-week treatment with the COX-2selective inhibitor effica-ciously downregulated P-glycoprotein to control levels(van Vliet et al.,2010).In phenobarbital resistant rats,a6-day celecoxib treatment was sufficient to overcome pharmaco-resistance(Schlichtiger et al.,2010).Therefore,these experimental data indicate that short phases of COX-2inhi-bition can normalize P-glycoprotein expression and func-tion at the blood–brain barrier,thereby improving antiepileptic drug brain penetration and efficacy.Conse-quently,some days of COX-2inhibitor add-on might help to restore pharmacosensitivity,and add-on treatment might be reestablished if seizure control worsens again.As a matter of course,short periods of COX-2inhibition will decrease the specific risks associated with COX-2inhibitors. Considering the adverse events associated with the use of COX-2inhibitors,we were eager to identify alternate tar-gets in the P-glycoprotein signaling cascade.Based on the practical therapeutic considerations outlined earlier we focused on the EP1receptor,which was confirmed as a downstream signaling factor mediating the effects on pros-taglandin E2on P-glycoprotein in endothelial cells.In the rat pilocarpine model,the EP1receptor antagonist SC-51089abolished seizure-induced P-glycoprotein upreg-ulation and retained its expression at control level(Fig.2) (Pekcec et al.,2009).Preliminary evidence was obtained that the approach will also improve antiepileptic drug effi-cacy.Targeting EP1receptors by selective antagonists might result in further beneficial effects in the epileptic brain.Experimental studies in rodent models of ischemic brain damage revealed that EP1receptors contribute to excitotoxicity and neuronal damage(Ahmad et al.,2006; Abe et al.,2009).In our study,we did not observe protec-tive effects of SC-51089on hilar neurons in the pilocarpine status epilepticus model(Pekcec et al.,2009).However,we did not perform a thorough evaluation of neuronal cell loss in other brain regions,and,therefore,cannot exclude protec-tive effects of SC-51089in other areas with seizure-associ-ated neuronal damage such as the cornu ammonis or the piriform cortex.Further studies are necessary to evaluate whether EP1receptor antagonism can mediate neuroprotec-tion in the epileptic brain.Regarding the potential to cause peripheral side effects,additional research is necessary. Therefore,it seems too early to draw conclusions about the risk–benefit ratio of EP1receptor antagonists as a putative add-on treatment in pharmacoresistant epilepsy. Regarding further translational development of the gen-eral approach,it is of critical relevance to exclude species differences in the regulatory mechanisms.Therefore,exper-iments are currently underway to analyze the role of the key signaling factors in human capillaries isolated from tissue dissected during epilepsy surgery.
Considering translational development,it is of specific interest that steroids with their antiinflammatory effects are already used in clinical practice.For several years,
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targeting brain inflammation has been suggested for sev-eral reasons,including an inhibition of pathophysiologic mechanisms that contribute to disease development or pro-gression(Vezzani&Granata,2005).Recently,Granata et al.(2009)intensely discussed the promises of antiin-flammatory drugs in the treatment of drug-resistant epi-lepsy.Steroids have been clinically used in epileptic syndromes including West syndrome,Lennox-Gastaut, Landau-Kleffner,continuous spike and waves during sleep, and Rasmussen’s encephalopathy as well as other forms of generalized and focal epilepsies(Granata et al.,2009). Efficacy was suggested by different studies in pediatric patients(Gupta&Appleton,2005;Verhelst et al.,2005; Araki et al.,2006;Granata et al.,2009).As discussed by (Granata et al.(2009),evidence exists that the beneficial effects might be linked to a modulation of the state and function of the blood–brain barrier.Interestingly,it has been described that blood–brain barrier damage and increased serum protein extravasation can be associated with a reduction in free phenytoin brain levels(Marchi et al.,2009a).Based on this study,blood–brain barrier leakage and resulting brain edema might reduce free con-centrations of antiepileptic drugs and may thereby contrib-ute to refractoriness.In another study,the same group has linked inflammatory reactions,blood–brain barrier dam-age,and seizure onset in a rat status epilepticus model (Marchi et al.,2009b).Therefore,taking available data into consideration,antiinflammatory strategies might increase free antiepileptic drug brain concentrations by two mechanisms,that is counteracting blood–brain barrier damage and controlling P-glycoprotein expression.
Comparison with Alternative Approaches to Overcome
Enhanced Ef?ux Transport Inhibiting P-glycoprotein transport function by adminis-tration of transporter modulators has been repeatedly suggested as an obvious approach to overcome transporter-associated pharmacoresistance(Table2).Experimental support came from studies indicating that P-glycoprotein modulation improves antiepileptic drug efficacy(Clinckers et al.,2005;van Vliet et al.,2006).Even more convincingly, add-on of the P-glycoprotein modulator tariquidar restored pharmacosensitivity in a chronic model of phenobarbital-resistant epilepsy(Brandt et al.,2006).Case reports are so far limited to the use of the nonselective P-glycoprotein mod-ulator verapamil(Summers et al.,2004;Iannetti et al.,2005, 2009).Success described in these reports needs to be consid-ered with caution for several reasons,including the low number of patients and the additional pharmacodynamic and pharmacokinetic effects of verapamil.Therefore,it has not been possible to draw conclusions so far about the clinical efficacy.
The major problem with the application of this approach is related to its intended aim,that is,reducing P-glycopro-tein function.Its application needs to take into account that the approach might not only target overexpressed P-glyco-protein but also P-glycoprotein expressed at normal basal levels exerting its protective function.At different biologic barriers,including the blood–brain barrier,P-glycoprotein serves as a gatekeeper that reduces exposure of tissues to harmful xenobiotics(Huls et al.,2009).In this context,it is of specific interest that a link between P-glycoprotein expression,pesticide exposure,and risk of Parkinson’s dis-ease is suggested by a series of studies(Lee&Bendayan, 2004).Parkinson’s disease results from the oxidative-stress–induced death of neurons in the substantia nigra region of the brain(Lai et al.,2002).Several pesticides, which are P-glycoprotein substrates,are capable of inducing oxidative stress and have been implicated in the pathophysi-ology of Parkinson’s disease in a subpopulation of patients (Lai et al.,2002).Interestingly,P-glycoprotein genotypes proved to be correlated with sensitivity to pesticide expo-sure and the resulting increased incidence of Parkinson’s disease(Furuno et al.,2002;Drozdzik et al.,2003;Lee et al.,2004;Tan et al.,2005;Zschiedrich et al.,2009).Fur-ther support for the suggested link comes from imaging studies reporting that brain penetration of the P-glycopro-tein substrate radiotracer[(11)C]verapamil or its(R)-enan-tiomer is elevated in patients with progressed Parkinson’s disease(Kortekaas et al.,2005;Bartels et al.,2008a,b). Moreover,ABCB1/MDR1transcripts encoding P-glycopro-tein are reduced in patients with Parkinson’s disease as com-pared to control individuals.Therefore,long-term inhibition of P-glycoprotein might predispose to Parkinson’s disease, especially in individuals with exposure to pesticides.The risk for development of other neurodegenerative disorders might be enhanced as well.Several studies indicated that P-glycoprotein expression and efflux function in blood–brain barrier endothelial cells is inversely correlated with the Abeta deposition(Vogelgesang et al.,2002;Cirrito et al.,2005).Therefore,it has been hypothesized that P-glycoprotein is a critical factor for Abeta clearance from the brain,and that low levels of P-glycoprotein transport activity are associated with an increased risk for Alzheimer’s disease.In addition to its function at the blood–brain barrier,the role in peripheral tissues and cells needs to be taken into consideration.P-glycoprotein serves transport functions in the liver,intestine,blood–testes barriers, kidney,placenta,and hematopoietic cells(Fromm,2004). Therefore,chronic inhibition of P-glycoprotein function might increase further disease risks.To give one example, evidence exists that low expression levels of P-glycoprotein predispose to chronic inflammatory bowel disease (Yamamoto-Furusho,2007).Therefore,clinical implemen-tation of P-glycoprotein modulators in pharmacoresistant epilepsies should consider different safety issues.One might argue for the use of nonselective P-glycoprotein modulators
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such as verapamil,for which clinical experience exists regarding long-term use in other indications.However,dos-ing might be limited by its additional pharmacodynamic effects,including reduction of heart rate and blood pressure (Iannetti et al.,2005).Consequently,efficacious modula-tion of P-glycoprotein at the blood–brain barrier of patients with epilepsy might require more selective and potent inhib-itors such as tariquidar.On the other hand,highly selective and potent inhibitors are likely to bias the protective func-tion of P-glycoprotein in a pronounced manner.One option might be intermittent treatment.Provided that seizure con-trol can be achieved with the add-on treatment of a P-glyco-protein modulator,the modulator might be withdrawn following some days or weeks as the P-glycoprotein induc-ing seizure activity is abolished and P-glycoprotein can return to control levels.However,the efficacy and tolerabil-ity of intermittent P-glycoprotein modulation still needs to be evaluated.
The fact that targeting the regulatory cascade of P-glycoprotein by COX-2inhibition or EP1receptor antagonism efficaciously keeps or brings P-glycoprotein expression to control level in epileptic rats(Pekcec et al., 2009;Zibell et al.,2009;van Vliet et al.,2010)(Tables1 and2)is of specific interest,as this novel approach seems to preserve the physiologically relevant transport function of P-glycoprotein.Therefore,the strategy offers particular advantages regarding tolerability issues related to P-glyco-protein’s protective function.The same advantage is evident as compared to RNA interference approaches downregulating P-glycoprotein.In apparent contrast to the selective intervention with seizure-associated regulatory mechanisms,RNA interference will unselectively affect P-glycoprotein expression throughout the body(Table2). Moreover,efficacious downregulation of blood–brain bar-rier P-glycoprotein seems to be difficult to achieve.We recently reported a significant decrease in blood–brain barrier P-glycoprotein in response to administration of the respective siRNA(Fuest et al.,2009).However,we did not consider the downregulation efficacy sufficient to affect the brain penetration of P-glycoprotein substrates in a relevant manner.Thus efforts are necessary to optimize efficacy and target delivery to the blood–brain barrier. Alternate strategies to overcome transporter-associated pharmacoresistance comprise different approaches to by-pass the blood–brain barrier(Table2).Intranasal admin-istration has been proposed as one option that might allow for brain targeting.Three pathways are generally postulated that a drug administered into the nasal cavity may follow. These routes include direct delivery to the brain,for exam-ple,along nerve sheaths,axonal transport along neurons, and entry into the blood from the nasal mucosa(Graff& Pollack,2005).To which extent a molecule passes along these different routes and to which extent it is thus indeed targeted to the central nervous system,critically depends on its chemical features and its formulation(Ugwoke et al.,2001).We reported that phenobarbital concentrations in cortical dialysates following intranasal administration of a mucoadhesive preparation exceeded those obtained follow-ing intravenous administration of the same dosage(Czapp et al.,2008).However,a recent meta-analysis of all pub-lished studies on intranasal administration of central ner-vous system drugs did not reveal any pharmacokinetic evidence supporting a claim that nasal administration of drugs in humans will result in an enhanced delivery to their target sites in the brain compared with intravenous adminis-tration of the same drug under similar dosing conditions (Merkus&van den Berg,2007).Therefore,it is currently questionable whether intranasal administration can be con-sidered as a means for efficacious by-passing of the blood–brain barrier.
Blood–brain barrier disruption by hypertonic solutions has successfully been used in cancer patients(Kroll et al., 1998).As a drawback,osmotic disruption of the blood–brain barrier affects the barrier function in a nonspecific manner,therefore,also enhancing brain penetration of harmful xenobiotics.Moreover,it has been demonstrated in animal experiments that blood–brain barrier opening and albumin extravasation can facilitate or even cause epilepto-genesis(Ivens et al.,2007;Tomkins et al.,2007;van Vliet et al.,2007a),thus ruling out the consideration of this approach in epilepsies and generally restricting its applica-tion to single administrations.Even more importantly, Marchi et al.(2009a)reported that the extent of acute blood–brain barrier opening and development of seizures correlates both in humans and in a pig model. Nanoparticle-based approaches,also referred to as the Trojan Horse strategy,have been suggested as an alternate option to optimize delivery and efficacy of antiepileptic drugs(Bennewitz&Saltzman,2009).As a major obstacle, the distribution into the brain is often limited with these sys-tems(Bennewitz&Saltzman,2009).Finally,intracerebral administration including intracerebroventricular or intrathe-cal infusion,implantation of controlled release systems,or convection-enhanced delivery might constitute an alternate approach(Huynh et al.,2006;de Boer&Gaillard,2007). However,its application as a routine therapeutic strategy is limited by its invasive nature.Moreover,low distribution rates in the brain parenchyma have repeatedly been described as a problem with the clinical application of this approach.
Future Perspectives:Current Approaches to De?ne the
Clinical Relevance of Transporter Overexpression Final conclusions about the future promise of the novel approach to target P-glycoprotein regulatory cascades and normalize P-glycoprotein expression obviously require
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further data on the clinical relevance of P-glycoprotein overexpression in pharmacoresistant epilepsies.
Although there is strong evidence that enhanced expres-sion rates of P-glycoprotein can characterize the blood–brain barrier of patients with pharmacoresistant epilepsy (Tishler et al.,1995;Dombrowski et al.,2001;Aronica et al.,2004;Sisodiya et al.,2006),the functional impact on antiepileptic drug brain penetration and efficacy in patients is discussed in a controversial manner.Moreover,P-glyco-protein overexpression might simply be considered as one facet of a multifactorial problem,which is likely to involve other factors such as intrinsic severity,alterations in target sites,upregulation of other transporters,and increases in extracellular protein concentrations.
Agreement exists that answers would be needed for the following questions to finally determine the clinical relevance of P-glycoprotein overexpression:(1)To what extent does P-glycoprotein overexpression in human cap-illaries limit antiepileptic drug penetration?(2)Which percentage of patients with pharmacoresistant epilepsy exhibits increased blood–brain barrier P-glycoprotein?
(3)Do P-glycoprotein expression rates in patients correlate with pharmacosensitivity?(4)Is it sufficient to overcome P-glycoprotein overexpression in order to restore pharmacosensitivity in a respective subgroup of patients?
As described earlier,testing in rodent models has ren-dered positive answers to the respective questions,thereby confirming the hypothesis that P-glycoprotein plays a major role in pharmacoresistance in the different models used.In the clinical setting it is of course more difficult to address these questions.Standard in vitro models could be used to evaluate which antiepileptic drugs are substrates of human P-glycoprotein.With these approaches one should consider that antiepileptic drugs are highly lipophi-lic and pass membranes by rapid diffusion.In addition, antiepileptic drug might rather be low to medium affinity substrates of P-glycoprotein.Antiepileptic drugs generally pass the blood–brain barrier,and are only affected when pathophysiologic mechanisms upregulate P-glycoprotein–expression brain-penetration rates.This has elegantly been substantiated by van Vliet et al.(2007b).In apparent con-trast,high-affinity substrates of P-glycoprotein,including several anticancer drugs,are efficaciously kept from the brain at basal expression levels(Schinkel,1999).There-fore,experimental approaches to evaluate the substrate characteristics of antiepileptic drugs require transport assays that are sufficiently sensitive to identify highly lipophilic as well as low-to-medium affinity P-glycopro-tein substrates.So far evidence has been reported that phenytoin,phenobarbital,lamotrigine,levetiracetam,topi-ramate,and an oxcarbazepine metabolite are substrates of human P-glycoprotein(Marchi et al.,2005;Cucullo et al., 2007;Luna-Tortos et al.,2008,2009).On the other hand, data argue against a transport of valproic acid and carbamazepine(Baltes et al.,2007;Luna-Tortos et al., 2008).In addition,transport of antiepileptic drugs has been evaluated in a human colon carcinoma cell line.In these in vitro assays no evidence was obtained that carbamazepine,vigabatrin,gabapentin,phenobarbital,or lamotrigine are substrates of P-glycoprotein(Owen et al., 2001;Crowe&Teoh,2006).Moreover,Rivers et al. (2008)reported that carbamazepine,valproic acid,phenyt-oin,lamotrigine,and primidone are not likely to be sub-strates of P-glycoprotein based on their investigations in breast and lung cancer cell lines.
With respect to additional studies in rodents as well as in cell lines expressing rodent P-glycoprotein,it still needs to be determined whether these data can be extrapolated to the human P-glycoprotein isoform.For a review of these data,readers are referred to Schmidt and Loscher (2005);Kwan and Brodie(2006);and Remy and Beck (2006).In view of the current state of knowledge and the controversial findings,further research is obviously neces-sary to determine the substrate characteristics of different antiepileptic drugs.Approaches using blood–brain barrier models generated from tissue dissected during epilepsy surgery may,in particular,help to address the question about the impact on antiepileptic drug brain-penetration rates.
Of course,evidence that some antiepileptic drugs are affected by the human P-glycoprotein isoform and that others are not substrates,needs to be considered when draw-ing conclusions about the future promises of any approach to modulate P-glycoprotein expression or function.Modu-lating P-glycoprotein might,therefore,only help to over-come resistance to selected antiepileptic drugs,but might not help to overcome multidrug resistance.More precise knowledge about substrate specificities is,therefore,crucial to guide the putative future application of respective diag-nostics as well as therapeutic strategies to selected patients. These studies also need to consider the impact of P-glyco-protein on metabolites.So far,transport has been reported only for the oxcarbazepine metabolite10,11-dihydro-10-hydroxy-5H-dibenzo(b,f)azepine-5-carboxamide(10-OHCBZ)(Marchi et al.,2005).
Regarding questions(2)Which percentage of patients with pharmacoresistant epilepsy exhibits increased blood–brain barrier P-glyco-protein?and(3)Do P-glycoprotein expression rates in patients correlate with pharmacosensi-tivity?,strong efforts are currently being made to develop and validate imaging techniques that allow analysis of P-glycoprotein efflux function at the blood–brain barrier of individual patients(Kannan et al.,2009).Different meth-ods of studying P-glycoprotein functionality and its modu-lation have been described.Single photon emission computer tomography(SPECT)is a technique that enables analysis of P-glycoprotein functionality using99mTc-Se-stamibi as tracer(Hendrikse,2000;Sharma,2004).How-ever,there is evidence that99mTc-Sestamibi is not only a
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substrate for P-glycoprotein but also for members of the multidrug resistance–associated protein(ABCC/MRP) family(Hendrikse,2000).Considering positron emission tomography(PET),a series of radiotracers including [11C]verapamil have been evaluated for the quantification of P-glycoprotein–mediated transport by PET(Elsinga et al.,2004).Based on the knowledge that the(R)-enantio-mer of verapamil is metabolized less in man and has a lower affinity for calcium channels,it has been suggested that this enantiomer is the better and safer candidate as PET tracer for measuring P-glycoprotein function in vivo (Luurtsema et al.,2003).Consequently,the first approach to imaging P-glycoprotein in a small group of epileptic patients used R-[11C]verapamil(Langer et al.,2007). This pilot–PET study uncovered some evidence for asym-metries in R-[11C]verapamil kinetics in homologous brain regions located ipsilateral and contralateral to the seizure focus.However,none of the differences reached statistical significance,a fact that might be attributed to the small sample size.Therefore,the completion of further,more comprehensive,studies is needed for definite conclusions. Moreover,ongoing development of PET probes might result in optimized tracers for P-glycoprotein imaging (Dorner et al.,2009;van Waarde et al.,2009).
As a relevant advancement,there is proof that the rever-sal of P-glycoprotein function can be monitored by PET. The first generation P-glycoprotein modulator cyclosporin A efficaciously increases[11C]verapamil brain accumula-tion(Hendrikse et al.,1998;Syvanen et al.,2008).More-over,reversal of P-glycoprotein function and enhancement of R-[11C]verapamil brain penetration can also be achieved using the more-specific third-generation P-glyco-protein inhibitor tariquidar,both at the rat and the human blood–brain barrier(Bankstahl et al.,2008b;Wagner et al., 2009).The successive PET scanning using R-[11C]verapa-mil with and without pretreatment with a P-glycoprotein inhibitor will probably allow an even more precise determination of P-glycoprotein function when analyzing the difference in the radiotracer brain kinetics between both scans.We recently suggested4-[18F]fluoro-N-[2-(1-(2-methoxyphenyl)-1piperazimyl]ethyl-N-2-pyridimyl-benzamide(p-[18F]MPPF)([18F]MPPF)as another P-glycoprotein substrate radiotracer(la Fougere et al., 2010).Subsequent[18F]MPPF PET scans with and with-out pretreatment with the P-glycoprotein modulator tariqui-dar allowed conclusions about P-glycoprotein transport function.Recently we demonstrated that the impact of tari-quidar on the brain kinetics of[18F]MPPF correlates with the pharmacosensitivity toward phenobarbital in a chronic rodent model of pharmacoresistant epilepsy,in which phar-macoresistance proved to be associated with P-glycoprotein overexpression(Bartmann et al.,unpublished data). Therefore,the PET imaging approach with combined use of a P-glycoprotein substrate radiotracer and a P-glycopro-tein modulator seems to be appropriate to noninvasively determine P-glycoprotein efflux function at the blood–brain barrier.Its application in patients with epilepsy might help to identify the number of patients exhibiting P-glycoprotein expression.Moreover,comparative studies between phar-macoresistant and pharmacosensitive patients should enable testing for a correlation between P-glycoprotein function and the pharmacoresponse.
Provided that imaging in epilepsy patients provides gen-eral support for a role of P-glycoprotein,respective imaging techniques can also guide patient selection for future clini-cal studies.In particular,PET imaging of P-glycoprotein function may allow individualized application of approaches to overcome P-glycoprotein–associated phar-macoresistance.
Conclusions
In conclusion,the elucidation of P-glycoprotein regula-tory pathways that are activated in response to seizures sug-gested several novel targets to overcome transporter-associated resistance in epilepsies.Further evaluation in acute and chronic rodent models demonstrated efficacy of the respective novel approaches with control of P-glycopro-tein expression rates,and increased antiepileptic drug brain penetration and efficacy.Most importantly,it has also been demonstrated that COX-2inhibition can help to overcome pharmacoresistance in a rat model.
Further development of the approach needs to consider tolerability issues specific to the different targets.
A conclusive assessment of the future perspectives of the novel therapeutic approach requires further evaluation of the clinical relevance of P-glycoprotein overexpression.In this context,progress is currently being made in the development of optimized imaging techniques determining P-glycoprotein transport function at the blood–brain barrier of individuals.Provided that functional relevance of P-glycoprotein overexpression can be substantiated clinically,the critical question remains whether it will be sufficient to overcome P-glycoprotein overexpression as one putative mechanism of a multifactorial problem.In this context,other mechanisms such as intrinsic disease severity, alterations in targets,brain edema,and further efflux trans-porters need to be taken into account.
Acknowledgments
The authors’own work was supported by a research grant from the Deut-sche Forschungsgemeinschaft(DFG PO681/4-1)and from the European Community’s Seventh Framework Programme under grant agreement no. 201380(EURIPIDES).
Disclosure
The author has no conflict of interest.I confirm that I have read the Jour-nal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.
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