Ricardo Viana Soares

PONS Pr Gérard

Dr Aloïse Mabondzo

Study of the Antiepileptics Transport through the Immature Blood-Brain Barrier

Keywords: BCRP -Breast cancer resistance protein BBB -Blood-Brain Barrier Blood-Brain Barrier, ABC Transporters, SLC Transporters, Brain Development, Pharmacoresistance in Epilepsy

Resistance to Antiepileptic Drugs (AEDs) has been a major concern in infantile epilepsies such as for example the Dravet Syndrome. One hypothesis concerning the pharmacoresistance in epilepsy is that a decreased delivery of these drugs to the brain may occur in relation to changes in the Blood-Brain Barrier (BBB) function. BBB exhibits ATP-binding cassette (ABC) and SoLute Carrier (SLC) transporters at the surface of endothelial cells that control the blood-brain transport.

Pharmacoresistance in epilepsy may be linked to changes in the functions of these transporters since some AEDs are substrates of the P-glycoprotein (P-gP) and Breast Cancer Resistance Protein (BCRP) transporters. The increased expression of efflux transporters in epileptogenic tissue and the identification of polymorphisms in the efflux transporters genes of resistant patients further support this potential relationship. The interaction of endothelial cells with astrocytes and neurons during brain development could change the pattern of transporters in the BBB. AEDs are also known as either inducers or inhibitors of drug metabolic enzymes and membrane transporters. Taken together, these facts led us to test the following hypothesis: 1) the developing BBB in immature animals presents a different pattern of transporters that could change AEDs disposition in the brain of immature subjects; and 2) the chronic pharmacotherapy used in infantile epilepsies such as the Dravet Syndrome may change the transporters phenotype of the BBB. Our work showed that the expression of P-gP and Abreviations ABC -Adenosine triphosphate binding cassette AC -Astrocyte AED -Antiepileptic drug Ang -Angiopoetin

BCRP increases during development as a function of age. We also showed the maturation of the BBB has an impact on brain disposition of the studied AEDs. We finally observed an increase in the expression of various ABC and SLC transporters induced by the pharmacotherapy of the Dravet Syndrome in immature animals. One of the drugs used, valproic acid, appeared nonetheless to reduce the efflux activity of P-gP in developing and adult animals, which was confirmed in an in-vitro model of the immature BBB. Taken together, these results demonstrated that the interaction between the developing BBB and the AEDs chronic treatment may lead to differences in brain disposition of the AEDs that may impact on the response to AEDs. Keywords: Pharmacoresistance, Antiepileptic drugs, Dravet syndrome, Blood-Brain Barrier, Efflux Transporters. La résistance aux médicaments antiépileptiques (MAEs) est un des problèmes majeurs des épilepsies infantiles, comme par exemple le syndrome de Dravet. La pharmacoresistance de l'épilepsie pourrait s'expliquer par une diminution du passage des MAEs dans le cerveau, à travers la Barrière Hémato-Encéphalique (BHE). La BHE comporte des transporteurs des familles « ATP-binding cassette » (ABC) et « SoLute Carrier » (SLC) localisés au niveau de la membrane des cellules endothéliales qui contrôlent leur passage entre le sang et le cerveau. La pharmacoresistance des épilepsies a été associée à ces transporteurs car des MAEs ont été identifiés comme substrats de transporteurs comme la glycoprotéine-P (P-gP) et la « Breast Cancer Resistance Protein » (BCRP). L'hypothèse de cette relation est confortée par l'observation de l'augmentation de l'expression de ces transporteurs d'efflux dans le foyer épileptogène et par l'identification des polymorphismes dans les gènes des transporteurs chez des patients pharmacorésistants. L'interaction au cours du développement cérébral entre les cellules endothéliales et les neurones et astrocytes pourrait modifier le profil des transporteurs de la BHE. Les MAEs sont aussi connus pour être soit des inducteurs, soit des inhibiteurs des enzymes du métabolisme des médicaments et des transporteurs membranaires. Ces données nous permettent de faire les hypothèses suivantes: 1) La BHE en développement présente un profil de transporteurs différent de la BHE mature qui pourrait modifier le passage des MAEs vers le cerveau ; et 2) le traitement chronique administré au cours du syndrome de Dravet pourrait changer le phénotype des transporteurs de la BHE en développement. Nous résultats ont montré que la P-gP et la BCRP augment leur expression au cours du développement. La maturation de la BHE a aussi un impact sur le passage des MAEs étudiés. Nous avons constaté une augmentation de l'expression des différents transporteurs ABC et SLC étudiés pendant le développement de la BHE, suite au traitement chronique avec la thérapie du Syndrome de Dravet. L'acide valproïque, un des MAEs utilisé dans ce traitement, diminue l'activité d'efflux de la P-gP chez les rats en développement et adultes, ce qui a été confirmé dans un modèle in-vitro de BHE immature. Ces résultats mettent en évidence l'interaction entre la BHE en développement et le traitement chronique par les MAEs peut modifier leur distribution au niveau du cerveau et la réponse aux MAEs.

Abstract (French)

Mots-clés: Pharmacorésistance, Médicaments Antiépileptiques, Syndrome de Dravet, Barrière Hémato-Encéphalique, Transporteurs d'Efflux. Table 1. Pediatric Epileptic Syndromes Table 2. Current AEDs and their main molecular targets Table 3. Current SLC families Table 4. Comparison between human and rat brain development.

List of Figures

Table 5. Impact of CBZ treatment on the mRNA levels of selected ABC and SLC transporters in isolated rat brain microvessels at post-natal days 14, 21 and 56. Table 6. Impact of the CBZ treatment on the efflux activity of P-gP and BCRP efflux transporters in the BBB at post-natal days 14, 21 and 56.

Dissemination

The results obtained in this work have been presented in international congresses and meetings and are the subject of articles submitted or to be submitted.

Introduction

Epilepsy is a disease of the central nervous system characterized by the occurrence of seizures which affects all ages and may have different causes. Antiepileptic Drugs (AEDs) have been developed, which act by different mechanisms to reduce the frequency of seizures. Nonetheless, one third of the patients do not respond to this therapy, especially children who represent 62% of the resistant patients. Dravet Syndrome is an example of infantile epilepsies that is frequently resistant to AEDs.

The causes of this resistance to antiepileptics are not well known. In this work we focused on the hypothesis that the pharmacoresistance in Epilepsy is related to reduced concentrations of the AEDs in the epileptic brain. This may be caused by the brain-blood interface, also named Blood-Brain Barrier (BBB). This structure is composed of cells from the brain parenchyma, such as pericytes astrocytes and neurons, and most importantly the endothelial cells of the brain capillaries. The BBB has special characteristics that may be responsible for the resistance to AEDs. Endothelial cells form tight junctions that reduce the permeability of this barrier. They express many types of transporters on their surfaces that control the passage of blood-borne molecules towards the brain.

The work presented in the present PhD thesis is composed of three main parts.

1 -An exhaustive review of the published in-vitro BBB models, based on the culture of endothelial cells, focused on the impact of brain maturation in these models;

2 -An experimental work on the ontogeny of the surface transporters belonging to the ATPbinding cassette (ABC) and SoLute Carrier (SLC) families of genes, in the microvessels of the developing rat brain;

3 -An experimental work where it was studied the impact of the AEDs used to treat the Dravet Syndrome on the efflux transporters present in the endothelial cells of the developing rat BBB. each of these three parts corresponding to a manuscript either already submitted or to be submitted for publication.

State of the Art

Child Epilepsies

Epileptic seizures and Epilepsy

Epilepsy is a disease of the Central Nervous System characterized by the existence of seizures which can affect all ages from newborns to the elderly. Seizures are related to the abnormal and excessive synchronous activity of neurons within neuronal networks, which can be localized in a small region of the brain cortex or spread out to both hemispheres. The ILAE defines an epileptic seizure as "a transient occurrence of signs and/or symptoms due to abnormal excessive or synchronous neuronal activity in the brain" [START_REF] Fisher | Epileptic seizures and epilepsy: definitions proposed by the International League Against Epilepsy (ILAE) and the International Bureau for Epilepsy (IBE)[END_REF], which are recurrent and transiently interrupt normal brain function. Nonetheless, the exhaustive mechanisms by which neurons develop the synchronous activity characteristic of seizures are still unknown.

Epilepsy is defined by the International League Against Epilepsy (ILAE) as "a disorder of the brain characterized by an enduring predisposition to generate epileptic seizures and by neurobiological, cognitive, psychological and social consequences of this condition. The definition of epilepsy requires the occurrence of at least one epileptic seizure." [START_REF] Fisher | Epileptic seizures and epilepsy: definitions proposed by the International League Against Epilepsy (ILAE) and the International Bureau for Epilepsy (IBE)[END_REF]. Epilepsy is characterized by criteria such as etiology, age of onset, seizure type, responsiveness to pharmacological treatment and other concomitant disorders [START_REF] Forsgren | The epidemiology of epilepsy in Europea systematic review[END_REF]. Specific associations of these criteria to specific types of epileptic seizures have been identified as "epilepsy syndromes" by the ILA [START_REF] Berg | Revised terminology and concepts for organization of seizures and epilepsies: report of the ILAE Commission on Classification and Terminology, 2005-2009[END_REF]; each syndrome carries its own diagnostic criteria, prognosis and drug response.

Seizures are the hallmark of epilepsy; however humans can have seizures without having epilepsy.

One good example for this is febrile seizures in children: they may appear during an age-related period (1 to 4 years) after an infectious insult when body temperature rises, but most often this is an isolated episode without further affecting the child normal development [START_REF] Offringa | Prophylactic drug management for febrile seizures in children[END_REF].

Therefore, these patients usually do not require any chronic drug treatment.

Seizures are electro-clinical events. They result in non voluntary clinical features which correspond to the forced activity of the subsequent cortical regions involved from the onset to the end of the discharge. The diagnosis of seizures is essentially made by the description of symptoms from the patient itself or from a witness. Electroencephalography (EEG), a technique by which the electric activity of the brain is routinely recorded using electrodes on the scalp, is mandatory to precisely identify the region(s) of the brain involved in the epileptic discharge. Between seizures, during the socalled interictal periods, EEG may be normal or show various patterns of spikes and/or slow waves, which may occasionally be specific for a given epilepsy syndrome.

Seizures are mainly classified as either being focal (also called partial) or generalized. Focal seizures arise from neuronal activity from a discrete region of the brain in only one hemisphere. Affected individuals may be completely conscious of their symptoms, even perceive them before the discharge becomes clinically apparent, a phenomenon called aura. Consciousness can be however impaired to a varying degree. Generalized seizures imply bilateral neuronal discharges. They may be primarily or secondarily generalized. In the latter they correspond to focal seizures which evolve to affect both hemispheres, whereas the hypersynchronous activity is supposed to involve the whole cortex from the onset in the former. Generalized seizures are mainly characterized by their motor signs: tonic, clonic, tonic-clonic, atonic, or myoclonic seizures, also called "convulsive" seizures. Alternatively, absences seizures are non-convulsive generalized seizures, characterized by sudden and seconds lasting impairments of conscience that present with typical electroencephalographic features [START_REF] Panayiotopoulos | Typical absence seizures and related epileptic syndromes: assessment of current state and directions for future research[END_REF].

Childhood Epilepsies

The occurrence of seizures can begin from the first year of life. These episodes are nonetheless different from the adults: seizures in the neonatal period may have unclear symptoms, being difficult to define even in electroencephalographic records. Later on, they may appear as clear events, occasionally age-specific, such as epileptic spasms [START_REF] Berg | Revised terminology and concepts for organization of seizures and epilepsies: report of the ILAE Commission on Classification and Terminology, 2005-2009[END_REF].

Beside the occasional seizures which are symptoms of many infectious or traumatic events occurring in childhood, but are not considered as epilepsies, pediatric epilepsies are more frequent than adult epilepsies. The incidence of epilepsy in the pediatric ages is higher than in adults. Estimations of incidence of epilepsy during the first year of life in the European population report between 153-256 new cases per 100.000 inhabitants per year. The incidence of epilepsy then decreases with age, with a minimum around 20-39 years-old individuals, to increase again after the 6 th decade of life [START_REF] Dulac | Early seizures: causal events or predisposition to adult epilepsy?[END_REF][START_REF] Forsgren | The epidemiology of epilepsy in Europea systematic review[END_REF].

Similarly, pediatric epilepsies are more various than adult epilepsies. Focal epilepsies (also called in therapeutic trials "epilepsies with partial onset seizures") represent about the half of pediatric epilepsies, whereas the proportion is higher in adulthood. In addition, a non-lesional form of focal epilepsy is frequent in children (benign epilepsy with centro-temporal spikes), but does not exist in adults. Among the remaining epilepsies, the majority can be classified into epilepsy syndromes, based on distinct signs and symptoms [START_REF] Chapman | Neonatal seizures: controversies and challenges in translating new therapies from the lab to the isolette[END_REF].

The classification of Pediatric Epileptic Syndromes has been recently updated by the ILAE classification commission [START_REF] Berg | Revised terminology and concepts for organization of seizures and epilepsies: report of the ILAE Commission on Classification and Terminology, 2005-2009[END_REF]. They are reported in Table 2 according to the period of brain development when the first clinical manifestations appear. This list includes very severe epileptic syndromes, such as Dravet syndrome, West syndrome or Lennox-Gastaut syndrome, but also syndromes with better prognosis such as childhood absence epilepsy. Syndromes are defined by the age at onset, type of seizures, electroclinical characteristics, etiology and comorbidities.

Table 1. Pediatric Epileptic Syndromes [START_REF] Berg | Revised terminology and concepts for organization of seizures and epilepsies: report of the ILAE Commission on Classification and Terminology, 2005-2009[END_REF], adapted (in bold the most frequent syndromes according to age). These syndromes have frequently well defined genetic causes, which develop their consequences at different times of brain development, or are related to malformations that occur in the brain during the in-utero development [START_REF] Dulac | Early seizures: causal events or predisposition to adult epilepsy?[END_REF]. Examples of genetic epilepsies are Dravet Syndrome (DS, mutations in genes SCN1A and SCN1B) or Tuberous Sclerosis associated seizures (mutations in genes TSC1 and TSC2) which are usually part of West syndrome, Lennox-Gastaut syndrome or focal epilepsy [START_REF] Curatolo | Neurological and neuropsychiatric aspects of tuberous sclerosis complex[END_REF].

Age Epileptic Syndrome

Neonatal

Treatment of Epilepsy

The management of patients with epilepsy is mainly performed by pharmacological treatment with Antiepileptic Drugs (AEDs). These drugs act through various mechanisms of action and most drugs act through more than one mechanism. The action of AEDs can be simplified as to inhibiting excitatory synapses, such as glutamatergic synapses, inducing inhibitory synapses such as gabaergic synapses, or modifications in ionic conductances. Such mechanisms include the inhibition of sodium and calcium channels, induction of -aminobutyric acid (GABA) chloride channels, inhibition of the metabolism of inhibitory neurotransmitters such as GABA, or inhibition of glutamate-dependent excitatory receptors. The main mechanisms of action of the AEDs are summarized in table 2.

Table 2. Current AEDs and their main molecular targets [START_REF] Camfield | Incidence of epilepsy in childhood and adolescence: a population-based study in Nova Scotia from 1977 to 1985[END_REF][START_REF] Perucca | An introduction to antiepileptic drugs[END_REF] The development of AEDs has led to a considerable list of options to treat seizures. However, many patients do not respond completely to treatment and other therapeutic approaches have been used to treat Epilepsy. Surgery is a treatment option in essentially two clinical settings: in focal epilepsy resistant to antiepileptic drugs and with a clear identified region where seizures develop, such is the case of cortical dysplasia or to some extent tuberous sclerosis; or when a complete brain hemisphere is injured whereas the other is not, e.g. Rasmussen Syndrome [START_REF] Ryvlin | Epilepsy surgery in children and adults[END_REF]. Another interesting non-pharmacological approach to treat epilepsy, in particular pediatric epilepsy, is the ketogenic diet.

This therapy consists on shifting the child food intake towards a higher consumption of fat and low consumption of carbohydrates. This leads to a high rate of formation of ketone bodies, which are important energy substrates for the developing brain, with significant improvement on seizure control (Neal et al., 2008). Furthermore, vagus nerve stimulation has been applied in some pharmacoresistant children who are not eligible for epilepsy surgery [START_REF] Joshi | Advanced treatments for childhood epilepsy: beyond antiseizure medications[END_REF].

Dravet Syndrome.

Dravet Syndrome (DS) was first described by Charlotte Dravet as Severe Myoclonic Epilepsy of Infancy (SMEI) in 1978 and then recognized as an epilepsy syndrome by ILAE and updated several times (Dravet, 2011;Dravet and Oguni, 2013). Its incidence in epileptic children is estimated to be between 3% and 7% [START_REF] Caraballo | Dravet syndrome: a study of 53 patients[END_REF].

Since the first descriptions of the syndrome already a genetic basis had been recognized, because about 50% of patients had familial antecedents of seizures, particularly febrile seizures [START_REF] Benlounis | Genetic predisposition to severe myoclonic epilepsy in infancy[END_REF]. It became apparent that a common genetic background existed between the patients with milder epilepsies such as generalized tonic-clonic seizures, febrile seizures and the more severe symptoms such as Dravet syndrome. This led to the creation of the Generalized Epilepsies with Febrile Seizures Plus (GEFS + ) assembling all these epileptic disorders [START_REF] Scheffer | Generalized epilepsy with febrile seizures plus. A genetic disorder with heterogeneous clinical phenotypes[END_REF].

DS was then included in this group of disorders as its most severe form after a major mutation on SCN1A gene had been identified in families with this syndrome [START_REF] Scheffer | Dravet syndrome or genetic (generalized) epilepsy with febrile seizures plus?[END_REF][START_REF] Singh | Severe myoclonic epilepsy of infancy: extended spectrum of GEFS+?[END_REF].

The clinical features of DS appear at about six months of age, when normally developing children initially present generalized long lasting febrile clonic or tonic-clonic seizures. Further non-febrile seizures will also as well as partial seizures such as hemiclonias, though in alternating body sides [START_REF] Scheffer | Diagnosis and long-term course of Dravet syndrome[END_REF]. Children usually present with a normal EEG at onset [START_REF] Caraballo | Dravet syndrome: a study of 53 patients[END_REF]. As the child further grows up, other types of seizures emerge, such as myoclonic seizures and atypical absences. These are accompanied by changes in EEG (e.g. spike and waves) [START_REF] Caraballo | Dravet syndrome: a study of 53 patients[END_REF]. Triggering factors for seizures are very common and consist mainly of temperature changes (fever, infection or hot baths) but also light or sound (Dravet, 2011). As the child grows older, seizure frequency tends to decrease and occur predominantly during sleep [START_REF] Guerrini | Dravet syndrome: the main issues[END_REF].

Further extending the neurological symptoms, DS children have a normal cognitive development rate until the second year of age, where it stagnates or even degrades so that children severely mentally retarded are frequent after the age of 6 years [START_REF] Guerrini | Dravet syndrome: the main issues[END_REF]. Main deficits include attention disorders, poor motor coordination and gait abnormalities (Dravet, 2011). In addition DS carries one of the highest mortality risk in pediatric epilepsy, due to accidents or SUDEP (Sudden Unexplained Death in Epilepsy) (Dravet and Oguni, 2013).

The major mutation associated with the DS was first identified in 2001: they were de novo mutations on the α-subunit Na V 1.1 (SCN1A gene), coding for missense, frameshift or truncation mutations [START_REF] Claes | De novo mutations in the sodium-channel gene SCN1A cause severe myoclonic epilepsy of infancy[END_REF][START_REF] Ohmori | Significant correlation of the SCN1A mutations and severe myoclonic epilepsy in infancy[END_REF]. Mutations on the same channel were also identified in GEFS+ patients [START_REF] Escayg | Mutations of SCN1A, encoding a neuronal sodium channel, in two families with GEFS+2[END_REF] as well as in β-subunit Na V β1 (encoded by gene SCN1B) of the voltage-dependent sodium channel [START_REF] Wallace | Febrile seizures and generalized epilepsy associated with a mutation in the Na+-channel beta1 subunit gene SCN1B[END_REF]. The mutations in SCN1A presented in a group of DS patients seemed to cause a loss of function of the voltage-gated sodium channel [START_REF] Sugawara | Nav1.1 channels with mutations of severe myoclonic epilepsy in infancy display attenuated currents[END_REF], whereas in GEFS+ patients, mutations apparently increased sodium channel function [START_REF] Lossin | Molecular basis of an inherited epilepsy[END_REF][START_REF] Rhodes | Sodium channel dysfunction in intractable childhood epilepsy with generalized tonic-clonic seizures[END_REF]. Further analysis identified mutations in about 80% of DS patients and cases of mosaicism in parents with SCN1A mutations [START_REF] Gennaro | Somatic and germline mosaicisms in severe myoclonic epilepsy of infancy[END_REF][START_REF] Morimoto | SCN1A mutation mosaicism in a family with severe myoclonic epilepsy in infancy[END_REF].

Since then, mutations in PCDH19, a gene coding for procadherine, have been found in 5% of the DS females negative for SCN1A [START_REF] Depienne | Sporadic infantile epileptic encephalopathy caused by mutations in PCDH19 resembles Dravet syndrome but mainly affects females[END_REF], so that PCDH19 is now considered as the second gene involved in DS. Mutations on the γ2-subunit of the GABA A receptor were also detected in few

Dravet patients [START_REF] Baulac | First genetic evidence of GABA(A) receptor dysfunction in epilepsy: a mutation in the gamma2-subunit gene[END_REF].

Pharmacological treatment of DS has been addressed in very few clinical trials. The mainstay of the treatment is the combination of VPA, STP and CLB [START_REF] Wilmshurst | Summary of recommendations for the management of infantile seizures: Task Force Report for the ILAE Commission of Pediatrics[END_REF]. STP is the only AED approved for treatment of DS after an exploratory open trial and two placebo-controlled randomized trials were conducted in French and Italian patients: these trials showed more than a 50% reduction in seizures frequency in about 70% of patients concomitantly treated with VPA and CLB (Chiron et al., 2000;[START_REF] Kassaï | Severe myoclonic epilepsy in infancy: a systematic review and a meta-analysis of individual patient data[END_REF][START_REF] Perez | Stiripentol: efficacy and tolerability in children with epilepsy[END_REF]. These results were further confirmed in observational studies on DS patients in Japan and US; moreover response to STP was observed even in patients who did not receive co-medication with VPA or CLB [START_REF] Inoue | Stiripentol open study in Japanese patients with Dravet syndrome[END_REF][START_REF] Inoue | Effectiveness of add-on stiripentol to clobazam and valproate in Japanese patients with Dravet syndrome: additional supportive evidence[END_REF]Wirrell, 2013). The mechanism of action of STP appears to be related not only to direct activation of GABA A receptors but also through a pharmacokinetic drug-drug inhibition of CLB metabolism (Quilichini et al., 2006).

TPM can also be useful to decrease seizure frequency in children with DS, but only observational studies were performed (Chiron, 2011). On the opposite, sodium channel blocker AEDs such as LTG, CBZ and PHT tend to actually exacerbate seizures in DS patients and are not recommended [START_REF] Wilmshurst | Summary of recommendations for the management of infantile seizures: Task Force Report for the ILAE Commission of Pediatrics[END_REF]. Finally, ketogenic diet has also been tried in DS with interesting results including in patients already treated with VPA, STP and CLB: 66% of the patients in ketogenic diet had a reduction in seizure frequency of at least 75% after 3 months of ketogenic diet [START_REF] Nabbout | Ketogenic diet also benefits Dravet syndrome patients receiving stiripentol: a prospective pilot study[END_REF]. Other authors also reported a reduction of at least 50% in seizure rate in 70% of the patients after 3 months of diet and 60% after one year [START_REF] Dressler | Efficacy and tolerability of the ketogenic diet in Dravet syndrome -Comparison with various standard antiepileptic drug regimen[END_REF].

DS is nonetheless still considered a pharmacoresistant syndrome. It has a very low probability to get completely control of the seizures and the likelihood of remission is also very low. DS summarizes the most significant predicting risk factors of pharmacoresistance: the early onset, high initial seizure frequency and the intellectual impairment (Wirrell, 2013). Furthermore, surgery is not suitable for DS children because DS does not present as focal epilepsy.

Focal Epilepsies

Focal (or partial) seizures are caused by the firing of neurons in a discreet area of the brain cortex. As previously mentioned, signs and symptoms which are displayed in seizures will vary depending on the affected region, and focal seizures may also spread locally or occasionally become secondarily generalized. The incidence of focal seizures in pediatrics is higher than generalized seizures: they account for 52% to 68% of the new cases, with an estimated incidence of 63.5 cases per 100000 persons each year [START_REF] Camfield | Incidence of epilepsy in childhood and adolescence: a population-based study in Nova Scotia from 1977 to 1985[END_REF]Wirrell, 2013).

Epilepsies with partial onset seizures are called focal epilepsies. Contrary to the other pediatric epilepsies which are mostly age-related, focal epilepsies may occur at any age from birth to adulthood, although occipital epilepsies usually occur earlier in life than frontal epilepsies, according to the sequential brain maturation [START_REF] Chiron | Treatment strategies[END_REF][START_REF] Chiron | Changes in regional cerebral blood flow during brain maturation in children and adolescents[END_REF]. Beside pure focal epilepsies with only focal seizures, several "non-determined whether focal or generalized" epilepsy syndromes have been described in pediatric patients, which associate focal and generalized seizures in an age-related profile [START_REF] Berg | Revised terminology and concepts for organization of seizures and epilepsies: report of the ILAE Commission on Classification and Terminology, 2005-2009[END_REF]. Among the most illustrating examples are West syndrome (or Infantile spasms), frequently associating focal seizures to epileptic spasms and an hypsarrhythmic EEG pattern in infants before 1 year of age [START_REF] Hrachovy | Infantile spasms[END_REF], and Lennox-Gastaut syndrome which associates focal seizures to generalized tonic, atonic and absence seizures with specific slow spike and waves on interictal EEG [START_REF] Arzimanoglou | Lennox-Gastaut syndrome: a consensus approach on diagnosis, assessment, management, and trial methodology[END_REF]. Focal epilepsy, West syndrome (Infantile spasms, IS) and Lennox-Gastaut syndrome can occasionally affect the same patient at different ages, especially in case of tuberous sclerosis (TS): TS is the most frequent cause of IS, focal seizures are most often associated with IS due to TS, and Lennox-Gastaut syndrome used to be the epilepsy outcome of the non-controlled IS due to TS [START_REF] Curatolo | Neurological and neuropsychiatric aspects of tuberous sclerosis complex[END_REF].

First line AED treatment of focal seizures still remains on the administration of CBZ in children in Europe, except under the age of 2 years where CBZ may worsen IS and is therefore not recommended as long as the proof of pure focal seizures has not been established [START_REF] Chiron | Treatment strategies[END_REF].

Contrary to adults, only OXC is approved for first line monotherapy among the new AEDs. Therefore second line most often consists in adjunctive therapy to CBZ, either clobazam or one of the new AED.

The choice among them mostly depends on age (versus AED approval), seizure frequency (versus titration schedule), and potential interactions (versus pharmacokinetic profile). OXC, LTG, GBP, TPM, LEV are currently approved in children over 4 years, perampanel also is approved over 12 years, but only LEV is approved in infants and lacosamide, pregabaline, retigabine, zonisamide and eslicarbazepine are only approved in adults to date. In TS, vigabatrin is the drug of choice because of its demonstrated efficacy as first line monotherapy in IS due to TS [START_REF] Chiron | Treatment strategies[END_REF][START_REF] Chiron | Randomized trial comparing vigabatrin and hydrocortisone in infantile spasms due to tuberous sclerosis[END_REF]. Due to the relationships between TSC1/TSC2, the TS genes, and the mTOR pathway, mTOR inhibitor everolimus has been approved for treatment of SEGA in children and is currently under trial for focal seizures in TS over 4 years of age [START_REF] Krueger | Everolimus long-term safety and efficacy in subependymal giant cell astrocytoma[END_REF] Cortical Dysplasia (FCD) as the major etiology. FCD is detected in up to 7.5% of patients submitted to epilepsy surgery, moreover this percentage can reach up to 26% when considering exclusively the pediatric population submitted to surgery [START_REF] Chassoux | Stereoelectroencephalography in focal cortical dysplasia: a 3D approach to delineating the dysplastic cortex[END_REF]. FCD appears to be responsible for higher seizure frequencies, younger onset, and a higher proportion of patients with daily seizures than other lesions [START_REF] Hauptman | Surgical treatment of epilepsy associated with cortical dysplasia: 2012 update[END_REF][START_REF] Semah | Is the underlying cause of epilepsy a major prognostic factor for recurrence?[END_REF].

In 1971 Taylor and colleagues identified a group of patients with intractable focal epilepsies submitted to brain surgery that presented marked histological disturbances in the removed tissue [START_REF] Taylor | Focal dysplasia of the cerebral cortex in epilepsy[END_REF]. These findings clearly distinguished this group of patients: although they presented seizures similar to tuberous sclerosis, the microscopic findings of the removed tissue suggested an independent etiology. FCD is characterized by the disruption of the normal neuronal laminar distribution in the cortical grey matter, with blurring of the typical clear frontier between grey and white matter [START_REF] Taylor | Focal dysplasia of the cerebral cortex in epilepsy[END_REF]. According to the ILAE updated classification, FCD type II remains the most typical form, with the classical giant dysmorphic neurons eventually associated to the balloon cells, whereas type I is less characteristic and often more extended. A new type III is now considered which dual pathology, that is FCD type II and remote hippocampal sclerosis, glial tumors, or other brain malformations [START_REF] Blümcke | The clinicopathologic spectrum of focal cortical dysplasias: a consensus classification proposed by an ad hoc Task Force of the ILAE Diagnostic Methods Commission[END_REF]. FCD type II is the perfect model of focal epilepsy since this lesion proved to be intrinsically epileptogenic: spontaneous seizures were directly recorded from the FCD using intracranial EEG with depth electrodes [START_REF] Chassoux | Stereoelectroencephalography in focal cortical dysplasia: a 3D approach to delineating the dysplastic cortex[END_REF] and lesion brain tissue from adult patients operated on from FCD showed long-lasting epileptiform activity when exposed to 4aminopyridine in an in vitro culture [START_REF] Mattia | Seizure-like discharges recorded in human dysplastic neocortex maintained in vitro[END_REF]. Although a proportion of patients up to 80% can reach seizure freedom after epilepsy surgery, there is still a proportion of patients that continue to have seizures after resection of the dysplastic area [START_REF] Dhamija | A population-based study of long-term outcome of epilepsy in childhood with a focal or hemispheric lesion on neuroimaging[END_REF].

Resistance to antiepileptic drugs

Resistance to AEDs is of currently importance problem in the management of epileptic disorders.

Despite the large number of AEDs that are available today to the neurologist and the development of consensus guidelines for the treatment of seizures, still a proportion of patients do not respond to the AEDs.

A practical definition of pharmacoresistant epilepsy has been proposed by the International League Against Epilepsy in 2010. Accordingly, "Drug resistant epilepsy may be defined as failure of adequate trials of two tolerated and appropriately chosen and used AED schedules (whether as monotherapies or in combination) to achieve sustained seizure freedom." [START_REF] Kwan | Definition of drug resistant epilepsy: consensus proposal by the ad hoc Task Force of the ILAE Commission on Therapeutic Strategies[END_REF].

The development of pharmacoresistance in epileptic patients can differ individually, with patients who never respond appropriately to any AEDs and other patients who may have a fluctuating pattern, with periods with lack of response to the therapy and other periods of time where seizures are controlled (Gorter and Potschka, 2012). About one third of the epilepsy patients treated with AEDs will never respond to treatment, and fewer that 50% will have a good response to the first AED [START_REF] Kwan | Epilepsy after the first drug fails: substitution or add-on?[END_REF]. This phenomenon is even more important in the pediatric population because after the failure of the first administered AED the probability of seizure is reduced to less than 30% (Ramos-Lizana et al., 2012). Moreover, 62% of the pharmacoresistant children are less than 4 years-old and this is associated with impaired cognitive and motor development (Ramos-Lizana et al., 2012). The main risk factors of pharmacoresistance in epilepsy population are early seizure onset (before 1 year of age), high number of seizures at onset, and the existence of a brain lesion [START_REF] Berg | Complete remission of childhood-onset epilepsy: stability and prediction over two decades[END_REF].

The exact mechanisms through which resistance to the AEDs appear are not thoroughly discovered.

The two main hypotheses that have been the subject of research in the past years are related to modifications in the targets of the AEDs and the reduced brain access to the AEDs. Recently a third hypothesis, related to the severity of the disease has been developed but more evidence is still needed.

Drug transport related mechanisms will be discussed in more detail.

Intrinsic epilepsy severity

The hypothesis that resistance to AEDs is linked to the severity of the epilepsy was founded on the observation that patients who have high frequency of seizures are more commonly resistant to drugs.

This phenomenon would be related to the reorganization of neuronal networks in the brain or changes in individual neurons that became very sensitive to triggering events and would not respond to the AEDs [START_REF] Rogawski | Intrinsic severity as a determinant of antiepileptic drug refractoriness[END_REF]. The actual mechanisms through which this resistance could developed are still speculative, and more information concerning the neurobiological factors that impact on seizure severity must be obtained to further develop this hypothesis [START_REF] Potschka | P-Glycoprotein-mediated efflux of phenobarbital, lamotrigine, and felbamate at the blood-brain barrier: evidence from microdialysis experiments in rats[END_REF].

Modifications on drug targets

The AEDs exert their effects by inhibiting or stimulating receptors in neurons. These receptors can be voltage-gated ionic channels, neurotransmitters receptors or enzymes involved in the metabolism of neurotransmitters. The hypothesis that resistance to AEDs was caused by modifications in the drug targets postulates that either intrinsic modifications (such as polymorphisms or other types of mutations) or acquired modifications (e.g. induced by drugs) are present in pharmacoresistant epilepsy.

Direct evidence of reduced antiepileptic efficacy was observed in sodium channels of temporal lobe epilepsy patients submitted to surgery. These channels are the major receptors of AEDs such as carbamazepine or phenytoin, and in these patients the pharmacoresistance was associated with a reduced capacity of carbamazepine to induce protective hyperpolarization in neurons (Gorter and Potschka, 2012). One study also revealed that in an animal model of epilepsy, alternative splicing of the rat sodium channel rNav1.2/rNav1.3 was observed after 4 hours induced status epilepticus [START_REF] Aronica | Induction of neonatal sodium channel II and III alpha-isoform mRNAs in neurons and microglia after status epilepticus in the rat hippocampus[END_REF]. The authors could not however analyze if there was a different sensibility to AEDs in this splicing variant.

GABA receptors are also interesting examples of target modifications that could be related to pharmacoresistant epilepsy. Apparently, the activation of these receptors causes a reduction in its sensibility towards benzodiazepines that could be related to internalization of these receptors (Gorter and Potschka, 2012). Moreover, GABA receptors are chloride channels that transport chloride down a concentration gradient: when chloride intracellular concentrations are lower than extracellular, the net movement of chloride is towards the cell cytoplasm inducing hyperpolarization. This mechanism is tightly regulated by other chloride channels, such as the NKCC1 or KCC2 channels that keep intracellular chloride concentrations lower than in the extracellular fluid [START_REF] Maa | Diuretics and epilepsy: will the past and present meet?[END_REF]. This chloride gradient is inverted during early stages of brain development and most importantly it was also observed in epileptic patients [START_REF] Dzhala | NKCC1 transporter facilitates seizures in the developing brain[END_REF]. Activation of the GABA receptor by benzodiazepines could lead to an actual outflow of chloride ions, causing depolarization of the neuron and induce seizures [START_REF] Ben-Ari | GABA: a pioneer transmitter that excites immature neurons and generates primitive oscillations[END_REF][START_REF] Yamada | Cl-uptake promoting depolarizing GABA actions in immature rat neocortical neurones is mediated by NKCC1[END_REF].

These examples corroborate to some extent the possibility that drug target modifications are responsible for resistance to antiepileptics. Nonetheless increasing evidence concerning the reduction in brain concentrations of the AEDs are more strongly in favor of the drug transport hypothesis.

Drug transport related mechanisms of resistance

The brain is protected from potentially toxic substances present in the blood by a unique structure, the Blood-Brain Barrier (BBB). This barrier controls the passage of substances towards the brain and could reduce the AEDs concentrations in the brain, causing pharmacoresistant epilepsy (Gorter and Potschka, 2012). Patients resistant to AEDs frequently do not respond to a broad range of drugs, which act in different targets on neurons, suggesting that the resistance mechanisms may be unspecific. The ATP-binding cassette (ABC) efflux transporters and certain Solute Carriers (SLC) transporters that are present in the BBB seem to be a good explanation because they can transport molecules that have very different chemical structures. Resistance to AEDs should be linked therefore to high levels of ABC transporters in the epileptic brain and AEDs should also be substrates of these transporters.

Alterations of the expression of efflux transporters expression in the epileptic brain

The hypothesis that the resistance to AEDs is caused by the BBB appeared after the study of drug resistance in cancer. The decrease in the efficacy of certain anticancer drugs led to the discovery of the efflux transporter P-glycoprotein (P-gP) [START_REF] Juliano | A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants[END_REF]. The first identification of higher P-gP gene expression in the capillaries of brain tissue of patients operated on for pharmacoresistant epilepsy led to the formulation of this hypothesis [START_REF] Tishler | MDR1 gene expression in brain of patients with medically intractable epilepsy[END_REF]. These results were later confirmed and more genes related to drug resistance, such as ABCC1, ABCC2 and ABCC5 were upregulated in brain specimens of temporal lobe epilepsy patients [START_REF] Dombrowski | Overexpression of multiple drug resistance genes in endothelial cells from patients with refractory epilepsy[END_REF]. It should be stressed that these clinical conditions are associated with severe resistant epilepsy: they are therefore the typical candidates for epilepsy surgery and good examples of pharmacoresistant epilepsy.

The upregulation of resistance genes in the brain of epilepsy patients was also found in other brain cells. Astrocytes showed increased immunoreactivity to P-gP in tissue from patients with temporal lobe epilepsy with hippocampal sclerosis and focal cortical dysplasia (Aronica et al., 2012). This expression was not only observed in astrocytes close to the capillary endothelial cells, but also in other astrocytes further away. The transporter BCRP (Breast Cancer Resistance Protein) has also been detected in endothelial cells, astrocytes and neurons (Aronica et al., 2012). This suggests that P-gP may have also an important role in protecting glial cells in pathological conditions. Moreover, an increase in P-gP and MRP1 immunoreactivity was observed in neurons of brain tissue from patients operated for focal cortical dysplasia [START_REF] Sisodiya | Drug resistance in epilepsy: expression of drug resistance proteins in common causes of refractory epilepsy[END_REF]. This immunoreactivity was not observed in healthy neurons. Apparently, this increased expression of efflux transporters in brain parenchyma and brain endothelial cells allows a coordinated elimination of eventual toxic substances from brain cells towards the cerebrospinal fluid and the blood.

The relationship between P-gP expression and decreased brain concentrations of AEDs was analyzed in patients treated with oxcarbazepine [START_REF] Marchi | A pilot study on brain-to-plasma partition of 10,11-dyhydro-10hydroxy-5H-dibenzo(b,f)azepine-5-carboxamide and MDR1 brain expression in epilepsy patients not responding to oxcarbazepine[END_REF]. Its active metabolite 10-hydroxy-10,11dihydrocarbazepine (10-OHCBZ) was quantified in the brain tissue of patients operated on for temporal lobe epilepsy. These concentrations were further correlated with the expression levels of P-gP (mRNA levels), where a negative correlation was observed: higher expression of P-gP correlated with low concentrations of 10-OHCBZ.

The study of the efflux activity of P-gP in the brain of epilepsy patients has recently been developed

with PET-scan studies. A pilot test using [ 11 C]-verapamil as a P-gP substrate showed that this radioligand had an apparent lower diffusion in the epileptogenic tissue of patients with temporal lobe epilepsy when compared with the non epileptogenic tissue of the same patient [START_REF] Langer | Pharmacoresistance in epilepsy: a pilot PET study with the P-glycoprotein substrate R-[(11)C]verapamil[END_REF].

These results where further confirmed in a recent study: [ ]-verapamil uptake into the brain was negatively correlated with seizure frequency, and inhibition of P-gP with tariquidar (recognized as P-gp inhibitor) was most effective in healthy controls, which suggests higher expression of P-gP in pharmacoresistant patients [START_REF] Feldmann | P-glycoprotein expression and function in patients with temporal lobe epilepsy: a case-control study[END_REF]. Moreover, in a subset of pharmacoresistant patients that were later submitted to surgery to remove the epileptogenic foci, immunostaining of P-gP was higher when [ 11 C]-verapamil brain uptake was lower. Overall, these results pointed out that the higher expression of P-gP in pharmacoresistant patients that could increase the AEDs brain efflux.

The association between pharmacoresistant epilepsy and higher expression of efflux transporters was analyzed in animal models of epilepsy. By inducing seizures in rodents, either by injection of epileptogenic substances such as kainate or pilocarpine, or inducing status epilepticus through electrical stimulation, the increased expression of P-gP, MRP1 and MRP2 was observed (Aronica et al., 2012;Löscher and Potschka, 2005;van Vliet et al., 2004). This increased expression was observed not only in endothelial cells, but also in brain parenchyma cells such as astrocytes or neurons (van [START_REF] Van Vliet | Expression of multidrug transporters MRP1, MRP2, and BCRP shortly after status epilepticus, during the latent period, and in chronic epileptic rats[END_REF]. Interestingly, in the epilepsy models induced by electrical stimulation, the higher expression of P-gP was associated with reduced brain concentration of phenytoin (van [START_REF] Van Vliet | Inhibition of the multidrug transporter P-glycoprotein improves seizure control in phenytoin-treated chronic epileptic rats[END_REF]. Furthermore, when regional brain concentrations of phenytoin were measured, higher concentrations were achieved in brain regions with lower expression of P-gP (van [START_REF] Van Vliet | Region-specific overexpression of P-glycoprotein at the blood-brain barrier affects brain uptake of phenytoin in epileptic rats[END_REF].

Whenever selective inhibition of P-gP was induced by treatment with tariquidar, the brain concentrations of either phenobarbital or phenytoin were increased [START_REF] Brandt | The multidrug transporter hypothesis of drug resistance in epilepsy: Proof-of-principle in a rat model of temporal lobe epilepsy[END_REF][START_REF] Van Vliet | Region-specific overexpression of P-glycoprotein at the blood-brain barrier affects brain uptake of phenytoin in epileptic rats[END_REF].

The link between pharmacoresistant epilepsy and increased efflux transporters expression led intuitively to the search of single nucleotide polymorphisms (SNP) in these transporters genes. These potential polymorphisms could be related to a reduction on the absorption, distribution of the AEDs, or an increase in the elimination of these drugs [START_REF] Löscher | The clinical impact of pharmacogenetics on the treatment of epilepsy[END_REF]. Polymorphisms on the ABCB1 gene were first analyzed in pharmacoresistant patients. The 3435C>T SNP, which was associated with increased expression and activity of P-gP, was more frequent in pharmacoresistant patient than in nonpharmacoresistant or control individuals (Siddiqui et al., 2003). Other polymorphisms in P-gP were also associated with increased resistance to AEDs, such as the 2677G>T or the 1236C>T, in fact the existence of haplotypes combining these three SNPs were discovered in resistant patients [START_REF] Löscher | The clinical impact of pharmacogenetics on the treatment of epilepsy[END_REF]. However, studies reported that there was a lack of association between these polymorphisms and the reduced activity of valproic acid, carbamazepine or phenytoin in pharmacoresistant patients [START_REF] Grover | Absence of a general association between ABCB1 genetic variants and response to antiepileptic drugs in epilepsy patients[END_REF][START_REF] Haerian | Lack of association of ABCB1 haplotypes on five loci with response to treatment in epilepsy[END_REF]. This may be related to the lack of evidence concerning the possibility of VPA and CBZ being substrates of P-gP. Moreover, other AEDs appear to be weak substrates of efflux transporters, therefore limiting the impact of these polymorphisms in the clinical efficacy of the studied AEDs [START_REF] Löscher | The clinical impact of pharmacogenetics on the treatment of epilepsy[END_REF]. However, in-vitro assays where made with the genes containing these SNP that were transfected in cells later used for transport studies with P-gP substrates. This study showed that some of these SNP either alone or in combinations (1236T-2677T-3435T, 1236T-3435T, or 2677A alone) had an impact on P-gP efflux activity when AEDs such as CBZ, PHT, PB or LTG were present [START_REF] Hung | Functional evaluation of polymorphisms in the human ABCB1 gene and the impact on clinical responses of antiepileptic drugs[END_REF]. Conflicting results have shown that in some group of patients SNP in the ABCB1 and ABCC2 genes seem to be associated with pharmacoresistance whether in other patients this association was not observed [START_REF] Bruhn | Polymorphisms of the drug transporters ABCB1, ABCG2, ABCC2 and ABCC3 and their impact on drug bioavailability and clinical relevance[END_REF][START_REF] Subenthiran | Linkage disequilibrium between polymorphisms of ABCB1 and ABCC2 to predict the treatment outcome of Malaysians with complex partial seizures on treatment with carbamazepine mono-therapy at the Kuala Lumpur Hospital[END_REF][START_REF] Ufer | Non-response to antiepileptic pharmacotherapy is associated with the ABCC2 -24C>T polymorphism in young and adult patients with epilepsy[END_REF]. Finally, exploratory studies in order to search for other SNPs were made in pharmacoresistant patients of the pediatric population. Several SNPs were identified in a group of patients from Mexican origin and significant associations were observed for SNPs in the ABCC2 gene (Escalante-Santiago et al., 2014).

Interactions of AEDs with the efflux transporters

To further substantiate that pharmacoresistant epilepsy is linked to the expression of efflux transporters in the BBB, the AEDs should be evaluated as substrates of these transporters. Many studies have analyzed the ability of the two major ABC transporters expressed in the BBB, P-gP and BCRP, to promote the efflux of AEDs.

Several major AEDs have been identified as P-gP substrates mostly in in-vitro assays using cells overexpressing the efflux transporters. Phenytoin, phenobarbital, lamotrigine and levetiracetam were all found to be effluxed by P-gP, although to a lesser extent when compared with a prototypical P-gP substrate, digoxin (Luna-Tortós et al., 2008). These results were obtained in equilibrium conditions in order to detected weaker P-gP substrates, and were confirmed by inhibition assays using specific P-gP inhibitors such as tariquidar or MK571. Carbamazepine was studied but it was not found any interaction with P-gP, although a previous study using microdialysis in mice showed that inhibition of P-gP increased brain concentrations of CBZ [START_REF] Potschka | P-glycoprotein and multidrug resistanceassociated protein are involved in the regulation of extracellular levels of the major antiepileptic drug carbamazepine in the brain[END_REF]. Studies concerning the intestinal absorption of CBZ in mice also found no effect of P-gP [START_REF] Fortuna | Evaluation of the permeability and P-glycoprotein efflux of carbamazepine and several derivatives across mouse small intestine by the Ussing chamber technique[END_REF]. By contrast, AEDs with similar structures such as oxcarbazepine, eslicarbazepine or licarbazepine were identified as P-gP substrates in two cell lines (MDCKII and LLC-MDR1 cells), showing that small differences in structure can change dramatically the interactions with efflux transporters (Zhang et al., 2011).

The major AEDs were also studied as potential substrates of BCRP. In in-vitro assays with cell lines overexpressing BCRP, it was not observed any interaction between this transporter and phenobarbital, phenytoin, ethosuximide, primidone, valproic acid, carbamazepine, clonazepam, or lamotrigine [START_REF] Cerveny | Lack of interactions between breast cancer resistance protein (bcrp/abcg2) and selected antiepileptic agents[END_REF]. These AEDs were also analyzed as potential BCRP inhibitors, by studying its effects on the efflux of BCRP substrates such as prazosin and mitoxantrone. Only valproic acid at high concentrations (500μM) induced a small but statistically significant reduction in BCRP. A study with knock-out rats for genes both Abcb1a/b and Abcg2 (P-gP and BCRP, respectively), found out that the absence of BCRP originated increases in brain concentrations of phenobarbital, clobazam, zonisamide, gabapentin and levetiracetam (Nakanishi et al., 2013). Other drugs such as topiramate and tiagabine showed to be influenced by both transporters, indicating an overlap in the selectivity of these transporters. Topiramate was also shown to be substrate of P-gP in one study using knock-out mice for Abcb1a gene [START_REF] Sills | Pglycoprotein-mediated efflux of antiepileptic drugs: preliminary studies in mdr1a knockout mice[END_REF].

Because other ABC transporters such as MRP1, MRP2 and MRP5 are overexpressed in epileptic tissue, cell-lines overexpressing these transporters were used to identify potential substrates amongst AEDs. Levetiracetam, valproic acid, carbamazepine, lamotrigine and phenobarbital were not shown to be substrates of these transporters [START_REF] Luna-Tortós | Evaluation of transport of common antiepileptic drugs by human multidrug resistance-associated proteins (MRP1, 2 and 5) that are overexpressed in pharmacoresistant epilepsy[END_REF]. However, the authors understate that this method can lead to false negative results, particularly with high permeability drugs that can cross easily biological membranes. For instance, in rats knockout for MRP2 phenytoin was shown to reach higher concentrations in the brain when compared with wild-type rats (Potschka et al., 2003).

Most of the studies have analyzed the interaction between AEDs and ABC transporters, and only a few have looked on the interaction with other types of transporters such as SLC carriers. However, the interaction of VPA with SLC transporters has been the subject of several studies. Initial studies showed that VPA influx towards the brain was higher than efflux [START_REF] Cornford | Blood-brain barrier transport of valproic acid[END_REF]. This was also the first study to find an asymmetry between blood-to-brain and brain-to-blood transport of a substance. VPA competitively inhibited the brain uptake of acetate in primary brain endothelial cells derived from capillaries of calves, putting forward the possibility that VPA transport is carrier mediated (Terasaki et al., 1991). Work from Adkinson and colleagues showed that transport of VPA through the BBB was dependent on organic anion transporters sensitive to probenecid and also to carriers of the monocarboxylic acids (Adkison and Shen, 1996;Adkison et al., 1994Adkison et al., , 1996)). These studies were essentially performed in rodents. Interestingly, studies comparing the effects of probenecid in the transport of VPA and E- 2 -VPA (a VPA metabolite) showed increased concentrations of both molecules in the brains of rabbits [START_REF] Scism | Effects of probenecid on brain-cerebrospinal fluid-blood distribution kinetics of E-Delta 2-valproic acid in rabbits[END_REF]. Overall, this study brought further evidence that VPA is effluxed from the brain through a transporter sensitive to probenecid.

In microvessels of temporal lobe epilepsy patients with hippocampal sclerosis it was revealed a decrease in the expression of the monocarboxylic acid transporter 1 (MCT1) (Lauritzen et al., 2011).

These samples were compared to autopsy specimens from individuals without epilepsy and to patients with temporal lobe epilepsy without hippocampal sclerosis. The authors claimed that this reduction on MCT1 transporter may reduce VPA concentrations in the epileptic foci. Furthermore, this reduction in MCT1 expression may be caused by the inflammation observed in the hippocampal tissue. The reduction in brain capillaries of the MCT1 transporter was also observed in induced animal models of temporal lobe epilepsy that was accompanied by increase in the expression of MCT1 in astrocytes (Lauritzen et al., 2012). This further suggests that the permeability of VPA in the epileptic tissue may be conditioned by this transporter in pharmacoresistant epilepsies. Moreover, an unidentified monocarboxylate transporter was shown to be important for the uptake of VPA in trophoblast cell lines [START_REF] Utoguchi | Carrier-mediated transport of valproic acid in BeWo cells, a human trophoblast cell line[END_REF].

The organic anion transporters were identified as carriers of VPA. The most important transporters, OAT1 and OAT3 transporters, belong to the Solute Carriers family (SLC transporters), specifically the SLC22 sub-family. They are involved in the excretion of xenobiotics in the proximal convoluted tubule of the kidney but their expression is also detected in the brain [START_REF] Burckhardt | Transport of organic anions across the basolateral membrane of proximal tubule cells[END_REF]. VPA was used as a competitive inhibitor of the common OAT substrate, p-aminohippurate, and specific cloning of the rat Oat1 transporter in Xenopus oocytes showed that Oat1 transported VPA (Sekine et al., 1997[START_REF] Sekine | The multispecific organic anion transporter (OAT) family. Pflüg[END_REF]. It was further shown that the mouse Oat2 transporter mediated the uptake of VPA in Xenopus oocytes overexpressing this transporter [START_REF] Scism | Probenecid-inhibitable efflux transport of valproic acid in the brain parenchymal cells of rabbits: a microdialysis study[END_REF]. Uptake of fluorescein, a substrate of the anion transporter present in the mitochondrial membrane was inhibited by VPA [START_REF] Kobayashi | Isolation, characterization and differential gene expression of multispecific organic anion transporter 2 in mice[END_REF]. Although VPA in humans is majorly eliminated through hepatic metabolism, these transporters could be important on VPA disposition in other tissues such as the brain.

The study of the interaction of VPA with the SLC transporters also showed that VPA can act as an inhibitor. The L-carnitine transporter OCTN2, expressed in the brush-border of the placental membrane, was inhibited by VPA [START_REF] Lahjouji | L-Carnitine transport in human placental brush-border membranes is mediated by the sodium-dependent organic cation transporter OCTN2[END_REF].

The interaction of VPA with ABC transporters seems to be less probable. Studies with VPA showed that P-gP, BCRP, MRP1, MRP2 and MRP5 do not transport VPA (Baltes et al., 2007;[START_REF] Cerveny | Lack of interactions between breast cancer resistance protein (bcrp/abcg2) and selected antiepileptic agents[END_REF][START_REF] Luna-Tortós | Evaluation of transport of common antiepileptic drugs by human multidrug resistance-associated proteins (MRP1, 2 and 5) that are overexpressed in pharmacoresistant epilepsy[END_REF]. One study in bovine brain capillary endothelial cells suggested that VPA was effluxed by MRPs using probenecid as a MRP inhibitor. Nonetheless the authors did not discard the potential effects of other organic anion transporters that might be inhibited by probenecid:

higher concentrations of probenecid actually decreased the uptake of VPA, suggesting other mechanisms regulating VPA transport through endothelial cells [START_REF] Gibbs | Valproic acid uptake by bovine brain microvessel endothelial cells: role of active efflux transport[END_REF].

VPA may however be important in the regulation of the ABC transporters expression and function.

VPA was shown to induce a 4-fold induction in the expression and function of P-gP in tumor cell lines and also in rat liver tissue [START_REF] Eyal | The antiepileptic and anticancer agent, valproic acid, induces P-glycoprotein in human tumour cell lines and in rat liver[END_REF]. This induction seemed to be dependent on the activation of nuclear receptors such as the constitutive androstane receptor (CAR) and the progesterone X receptor (PXR) (Cerveny et al., 2007). VPA also appeared to induce P-gP expression in primary cultures of rat astrocytes [START_REF] Lü | Antiepileptic drug-induced multidrug resistance Pglycoprotein overexpression in astrocytes cultured from rat brains[END_REF].

The data concerning the drug transport hypothesis showed that it may potentially be the answer to the problem of pharmacoresistance in epilepsy. Not only should this information be useful to counteract the diminished activity of the AEDs but can also reveal new targets for the development of newer AEDs. Particularly to this work, the lack of information concerning transport of AEDs in the children should be the focus of future work in order to prevent the deleterious effects of uncontrolled seizures in the pediatric population.

Blood-Brain Barrier Drug Transport

The normal function of the central nervous system (CNS) necessitates a controlled environment surrounding the nerve cells. This control is achieved not only through a mechanical protection, such as the existence of skull or the meninges that protect the brain against physical aggression, but also the strict control of solutes concentration in the brain parenchyma in order to allow the normal function of the brain cells. Because the blood furnishes all the nutrients to the CNS, a selective interface was developed between the brain and the blood in animals with well developed CNS. This interface, the Blood-Brain Barrier, maintains the homeostasis of the extracellular fluid of the brain by selective uptake of nutrients from the blood but also the efflux of metabolic waste and potentially toxic xenobiotics that are blood-borne. The BBB is a cellular barrier: it is composed of endothelial cells lining brain capillaries, surrounded by a basement membrane were pericytes are present, that are in an intimate contact with brain parenchyma cells such as astrocytes and neurons. Other barriers exist in brain, such as the blood-cerebrospinal fluid barrier or the arachnoid barrier (Abbott et al., 2010a).

The BBB presents special features mainly observed in endothelial cells: lack of fenestrations, the expression of transporter proteins and metabolizing enzymes in endothelial cells, or a reduced number of vesicles. All these features can reduce the transport of substances between the blood and the brain, in particular the intentionally administered drugs to treat neurological disorders. Therefore, the BBB is a most important aspect of brain delivery, for instance as discussed above, for the study of resistance to the AEDs.

The role of the BBB on the pharmacoresistance to AEDs becomes even more complex when we consider childhood epilepsies. The developing brain may present a BBB that potentially has a different permeability when compared with the adult brain. This potential different permeability may be the consequence of a different pattern of transport mechanisms, for example a different pattern of transporter proteins.

Blood Brain Barrier

The BBB is composed primarily of the endothelial cells that shape the brain capillarie (Abbott et al., 2010a). These cells form an interface between the brain parenchyma and the circulation that represents a total are between 12 and 18 m 2 (Abbott et al., 2010a). The hallmark of these endothelial cells is the expression of tight junctions in between cells: this feature greatly reduces the passive diffusion of substances between the endothelial cells, the paracellular transport. These tight junctions stand for the physical barrier, allowing the endothelial cells to further control more accurately the passage of substances through the cytoplasm, the transcellular transport (Wolburg et al., 2009).

The tight junctions are composed of different proteins. Transmembrane proteins from one cell interact with other proteins on the adjacent cell allowing the adhesion between cells, forming a complex connection of transmembrane and cytoplasmatic strands of these proteins. The major proteins are Junctional Adhesion Molecules (JAM), the claudin family and occludin proteins (Cardoso et al., 2010a). Claudins 1, 3 and 5 appear to be the most important proteins in tight junctions of the BBB, while occludin appear to have a more supportive role (Cardoso et al., 2010b;Wolburg et al., 2009).

The intracellular domains of these proteins are in direct contact with other proteins, such as Zona Occludens (ZO) proteins, cingulin, 7H6 and AF-6. The actin cytoskeleton is linked with these cytoplasmatic proteins, which reinforces the tight junction within the whole cell (Abbott et al., 2010a).

Endothelial cells also present adherens junctions, mainly composed of cadherins. These junctions are connected with intracellular proteins named catenins (,  and  catenins) which are also linked to the cell actin cytoskeleton [START_REF] Ballabh | The blood-brain barrier: an overview: structure, regulation, and clinical implications[END_REF]. These junctions between brain endothelial cells reduce the paracellular transport through the BBB.

Pericytes are cells with smooth-muscle phenotype are present in the BBB and enveloped by the basement membrane (Liebner et al., 2011a). Their exact precursor is unknown and they appear to have an important role in secreting the basement membrane and also in the maintenance of the BBB (Cardoso et al., 2010b;Krueger and Bechmann, 2010). These cells are well known as regulators of the BBB function.

The entire length of the brain capillaries seem to be covered by specialized end-feet from astrocytes.

These cells appear to be involved in the maintenance of the BBB properties although they do not make part of the physical barrier. Astrocytes are also in close contact with neurons and can therefore be important in mediating the interactions between all cells that make part of the BBB (Cardoso et al., 2010b;[START_REF] Nedergaard | New roles for astrocytes: redefining the functional architecture of the brain[END_REF]. The differentiation of endothelial cells during BBB development is also closely linked to the differentiation of astrocytes (Wolburg et al., 2009).

Astrocytic endfeet when in contact with the endothelial cells develop orthogonal arrays of particles that are rich in aquaporins, a type of transmembrane protein which facilitates water movements (Liebner et al., 2011b). They appear to have a role in controlling regional blood-flow and also the osmotic environment of the BBB [START_REF] Wolburg | Structure and functions of aquaporin-4-based orthogonal arrays of particles[END_REF]. Co-culture of endothelial cells with astrocytes or with astrocytes-conditioned medium leads to an increase in tight junction complexity and reduced paracellular transport [START_REF] Wolburg | Modulation of tight junction structure in blood-brain barrier endothelial cells. Effects of tissue culture, second messengers and cocultured astrocytes[END_REF]. These results seem in part be caused by the expression of src-suppressed C-kinase substrate (SSeCKS) by astrocytes, who reduces the expression of vascular endothelial growth factor (VEGF) and induces angiopetin-1 expression [START_REF] Lee | SSeCKS regulates angiogenesis and tight junction formation in blood-brain barrier[END_REF].

Types of Transport

The existence of very tight junctions between the endothelial cells reduces the paracellular transport, restricting the entry of molecules to the brain through the endothelial cell.

The possible pathways for a drug to enter the brain are shown in Figure 1. The transport of a given molecule will depend on its structure, which is related to its size and its lipophilicity (measured as the octanol/water partition coefficient), and the possibility of interactions with membrane transporters or cell surface receptors. A simple rule of thumb is that small lipophilic molecules with a molecular weight inferior to 450 daltons (Da) will diffuse passively through the membrane of the endothelial cell (Abbott et al., 2010b). This is the case of many drugs that have their targets on the CNS, such as a large number of AEDs or general anesthetics. The extent of protein bonding is also very important for the diffusion of drugs to brain: the affinity to plasma or brain proteins may modulate the permeability of drug because only the free fraction (not bond to protein) can diffuse through the endothelial cell [START_REF] Kalvass | Use of plasma and brain unbound fractions to assess the extent of brain distribution of 34 drugs: comparison of unbound concentration ratios to in vivo p-glycoprotein efflux ratios[END_REF]. An apparent reduction in theophyline brain uptake in the adult rat when compared with neonatal was considered to be caused by higher protein content in the adult blood [START_REF] Habgood | Permeability of the developing and mature blood-brain barriers to theophylline in rats[END_REF].

The molecules that diffuse to the endothelial cell can however be captured by transporters in endothelial cell membrane, the ABC efflux transporters, reducing their passage to the brain. The ABC transporters efflux is an active mechanism eliminating substances against a concentration gradient and is therefore dependent on the hydrolysis of ATP. The normal function of the brain is however dependent of other blood-born molecules that have very low lipophlicity. This is the case nutrients such as glucose, nucleosides or amino acids that could therefore have a very low BBB permeability. For instance aminoacids are ionized at physiological pH, which reduces considerably its diffusion through the lipid bilayer of the cell membrane. In order to furnish these molecules to the brain, endothelial cells express a large number of carriers which belong to the SLC family. This family comprehends many transporters which can be energy dependent in the case of transport up a concentration gradient, or simple facilitators of the diffusion of molecules down a concentration gradient [START_REF] Hediger | The ABCs of solute carriers: physiological, pathological and therapeutic implications of human membrane transport proteinsIntroduction[END_REF]. Figure 2 presents the types of SLC transporters that can be exchangers, coupled transporters or passive transporters. Exchangers and coupled transporters frequently use electrochemical gradients such as those created by Na + /K + ATPase to facilitate the transport of molecules.

Molecules with a large molecular weight such as proteins are not expected to diffuse passively across the BBB. There a few examples of proteins, such as insulin or pituitary adenylate cyclase-activating polypeptide 27 that can be substrates of membrane transporters [START_REF] Banks | The source of cerebral insulin[END_REF][START_REF] Dogrukol-Ak | Isolation of peptide transport system-6 from brain endothelial cells: therapeutic effects with antisense inhibition in Alzheimer and stroke models[END_REF]. The majority of proteins cross the BBB through receptor -mediated transcytosis: the bloodborne protein activates a specific receptor in the apical membrane of the endothelial cell triggering the formation of a vesicle that transports the protein and the receptor towards the basolateral membrane where exocytosis occurs (Abbott et al., 2010a;Cardoso et al., 2010b). This transport may be also be elicited by non-receptor mechanisms rather the adsorption of the macromolecule to endothelial membrane elicits the formation of the vesicles (Abbott et al., 2010a). The occurrence of infections or inflammatory events in the brain parenchyma elicits the recruitment of immune cells from the general circulation. In the normal brain, the passage of immune cells towards the brain appears to occur through a transcellular pathway, where the entire immune cell is enclosed in a vesicle and exocytosed in the basolateral membrane (Abbott et al., 2010a). However, in pathological conditions this passage may be done by disruption of tight junctions, which are induced by perivascular macrophages and microglia [START_REF] Ortiz | Role of the blood-brain barrier in multiple sclerosis[END_REF]. In either situation, interaction between surface receptors of the immune cell and the endothelial cell, such as Vascular Cell Adhesion Molecules (VCAM) or Platelet Endothelial Cell Adhesion Molecules (PECAM), allows the circulating immune cell to slow down and more ligand-receptor interactions are established to induce transcytosis.

The marketed drug natalizumab, a humanized monoclonal antibody used in the treatment of multiple sclerosis, acts by inhibiting the 4-integrin on the surface of white cells, a molecule that is necessary for the interaction with VCAM in the endothelial cell, further inhibiting the passage of white cells towards the brain [START_REF] Ortiz | Role of the blood-brain barrier in multiple sclerosis[END_REF].

Membrane Transporters

The membrane of the brain endothelial cells present surface transporters that control the passage of diverse molecules across the BBB. The most important transporters belong to the ABC and the SLC families. While some transporters have a wide range of substrates, such as the most important ABC transporter P-gP, others are responsible for the transport of only one o very few molecules, such as the SLC glucose transporter GLUT-1. ABC transporters in the BBB appear to be linked to the detoxification of the brain, by removing potentially toxic xenobiotics and metabolites (Abbott et al., 2010). Although there are SLC transporters that are responsible for the elimination of xenobiotics, such as the organic anion transporters, much more are responsible for the influx of nutrients to the brain. As discussed above, some of these transporters have been implicated in pharmacoresistance to

AEDs by reducing their passage through the BBB (Löscher and Potschka, 2005).

ATP-binding cassette Efflux Transporters

The Adenosine triphosphate Binding Cassette (ABC) efflux transporters family consists of about 50 transmembrane proteins that utilize the energy obtained by hydrolysis of ATP to efflux molecules from the cell cytoplasm. Their structure is composed typically by two transmembrane domains and two nucleotide binding domains in contact with the cell cytoplasm. The transmembrane domains consist of up to 11 -helices that are responsible for substrate specificity [START_REF] Dean | The human ATP-binding cassette (ABC) transporter superfamily[END_REF]. This large family is divided in 8 subfamilies, ABCA till ABCG (Abbott et al., 2010a). The major efflux transporters that are present in BBB belong to family B, in particular ABCB1 or P-gP; family C, in particular the MRPs coded from genes ABCC1-6; and family G, whose major transporter is BCRP coded from gene ABCG2 (Abbott et al., 2010a). Resistance to drugs has been essentially related to these genes [START_REF] Dean | The human ATP-binding cassette (ABC) transporter superfamily[END_REF]. The first ABC transporter identified as related to drug resistance in tumors was P-gP [START_REF] Juliano | A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants[END_REF].

Solute Carriers Transporters

The Solute Carriers (SLC) make part of a very large family of transporters that encode for passive transport channels, ion coupled transporters or ion exchangers [START_REF] Hediger | The ABCs of solute carriers: physiological, pathological and therapeutic implications of human membrane transport proteinsIntroduction[END_REF]. A list of currently approved SLC families can be consulted in table 3. Some of these transporters have been identified as efflux transporters, which have been detected in the BBB [START_REF] Kusuhara | Active efflux across the blood-brain barrier: role of the solute carrier family[END_REF].

Table 3. Current SLC families [START_REF] Hediger | The ABCs of solute carriers: physiological, pathological and therapeutic implications of human membrane transport proteinsIntroduction[END_REF].

SLC family Description Members

SLC1

The high-affinity glutamate and neutral amino acid transporter family SLC2

The facilitative GLUT transporter family 14 SLC3

The heavy subunits of the heteromeric amino acid transporters SLC4

The bicarbonate transporter family 10 SLC5

The sodium glucose cotransporter family SLC6

The sodium-and chloride-dependent neurotransmitter transporter family 16 SLC7

The cationic amino acid/glycoprotein-associated amino acid transporter family 14 SLC8

The Na+/Ca2+ exchanger family SLC9

The Na+/H+ exchanger family SLC10

The sodium bile salt cotransport family 6 SLC11

The proton coupled metal ion transporter family SLC12

The electroneutral cation Cl cotransporter family SLC13

The human Na+-sulfate/carboxylate cotransporter family SLC14

The urea transporter family SLC15

The proton oligopeptide cotransporter family SLC16

The monocarboxylate transporter family 14 14 SLC17

The vesicular glutamate transporter family SLC18

The vesicular amine transporter family SLC19

The folate/thiamine transporter family SLC20

The type-III Na+-phosphate cotransporter family SLC21

The organic anion transporting family 11 SLC22

The organic cation/anion/zwitterion transporter family SLC23

The Na+-dependent ascorbic acid transporter family SLC24

The Na+/(Ca2+-K+) exchanger family SLC25

The mitochondrial carrier family 27 SLC26

The multifunctional anion exchanger family 10 SLC27

The fatty acid transport protein family SLC28

The Na+-coupled nucleoside transport family SLC29

The facilitative nucleoside transporter family SLC30

The zinc efflux family SLC31

The copper transporter family SLC32

The vesicular inhibitory amino acid transporter family SLC33

The acetyl-CoA transporter family SLC34

The type-II Na+-phosphate cotransporter family SLC35

The nucleoside-sugar transporter family 17 SLC36

The proton-coupled amino acid transporter family SLC37

The sugar-phosphate/phosphate exchanger family SLC38

The System A and N, sodium-coupled neutral amino acid transporter family SLC39

The metal ion transporter family 14 SLC40

The basolateral iron transporter family SLC41

The MgtE-like magnesium transporter family SLC42

The Rh ammonium transporter family (pending) SLC43

The Na+-independent, system-L-like amino acid transporter family Among these families, SLC21 (also known as SLCO), the organic anion transporting (OATP)

superfamily and SLC22, the organic cation/anion/zwitterion transporter family, are involved in the uptake of many diverse substrates. SLCO and SLC22 exhibit wide tissue distribution and have been identified in the BBB [START_REF] Dobson | Carrier-mediated cellular uptake of pharmaceutical drugs: an exception or the rule?[END_REF]. They appear to cooperate with ABC transporters in the efflux of xenobiotics (Deli and Joó, 1996;[START_REF] Tsuji | Small molecular drug transfer across the blood-brain barrier via carrier-mediated transport systems[END_REF]. Moreover, the SLC16 member 1, coding to the

Mct1 transporter, has been found to interact with the AED valproic acid [START_REF] Tsuji | Small molecular drug transfer across the blood-brain barrier via carrier-mediated transport systems[END_REF].

2.3.2.2.

Transporters Modulation

The expression and function of the efflux transporters is regulated in both transcriptional and posttranscriptional levels [START_REF] Miller | Regulation of P-glycoprotein and other ABC drug transporters at the blood-brain barrier[END_REF](Miller, , 2015)). Considering the regulation of the transcription of these genes, the transcription factor constitutive androstane receptor (CAR) was observed to be an inducer of P-gP, Mrp2 and Bcrp in rat brain microvessels (Wang et al., 2010). The authors used the AED phenobarbital as a CAR activator. More evidence exists for another transcription factor, the pregnane X receptor (PXR). Activators of this transcription factor such as rifampicine or hyperforin induced P-gP and Mrp2 [START_REF] Bauer | In vivo activation of human pregnane X receptor tightens the blood-brain barrier to methadone through P-glycoprotein up-regulation[END_REF][START_REF] Bauer | Coordinated nuclear receptor regulation of the efflux transporter, Mrp2, and the phase-II metabolizing enzyme, GSTpi, at the blood-brain barrier[END_REF]. Methadone, a P-gP substrate was, had lower brain permeability after activation of PXR [START_REF] Bauer | Pregnane X receptor up-regulation of P-glycoprotein expression and transport function at the blood-brain barrier[END_REF].

Blood-Brain Barrier Development

The BBB is a structure that starts to form in the early stages of the development of the CNS in the fetus. Nutrient supply during embryogenesis and early fetus development is mainly achieved by diffusion from closer structures of the embryo. The study of the development of the brain vasculature has been made with animal models, specially the mouse.

The formation of the brain parenchyma vasculature occurs only through angiogenesis (Figure 3) (Liebner et al., 2011b). Following the formation of the perineural vascular plexus that covers the pial surface of the embryo brain, endothelial cells start to sprout towards the interior of the brain through a radial and centripetal fashion in the mouse embryonic day 9 (Fig. 4) [START_REF] Fantin | The embryonic mouse hindbrain as a qualitative and quantitative model for studying the molecular and cellular mechanisms of angiogenesis[END_REF]Liebner et al., 2011b). Later, perpendicular sprouting of these radial vessels start to create a network of capillaries that when anastomose create the subventricular vascular plexus [START_REF] Fantin | The embryonic mouse hindbrain as a qualitative and quantitative model for studying the molecular and cellular mechanisms of angiogenesis[END_REF]. The sprouting and division of the endothelial cells is dependent on the secretion of paracrine factors by neuropercursor cells. Endothelial cells at this phase are guided by a gradient of vascular endothelial growth factor (VEGF) that act on neuropilin receptor 1 (NPR1) present on the endothelial cells, which induces the filopodia-like phenotype in the endothelial cells [START_REF] Fantin | The embryonic mouse hindbrain as a qualitative and quantitative model for studying the molecular and cellular mechanisms of angiogenesis[END_REF].

These initially formed brain capillaries start to differentiate to a proper BBB phenotype by increasing the interaction with the other cells that form the neurovascular unit (NVU). The expression of the platelet-derived growth factor (PDGF) induces the proliferation of pericytes along the capillaries, providing the formation of the basement membrane surrounding the NVU (Paolinelli et al., 2011).

Expression of leukemia inhibitory factor by the endothelial cells induces the differentiation of the astrocytes in the NVU, which in turn secrete other paracrine factors that induce the mature BBB phenotype in the endothelial cell (Liebner et al., 2011b). The src-suppressed C-kinase substrate

(SSeCKS) appears to be produced by astrocytes and it induces the canonical Wnt/-catenin pathway and angiopoetin-1 (Ang-1) expression [START_REF] Lee | SSeCKS regulates angiogenesis and tight junction formation in blood-brain barrier[END_REF]. Ang-1 and VEGF appear to have opposing effects: while VEGF induces a proliferative state in endothelial cells, destabilizing tight junctions and inducing endothelial cell division, Ang-1 promotes the differentiation of endothelial cells to a BBB phenotype (Thurston, 2002;[START_REF] Thurston | Angiopoietin-1 protects the adult vasculature against plasma leakage[END_REF]. The interaction of Wnt ligands such as Wnt7 a and b with the endothelial cell frizzled receptor activates the -catenin pathway: this promotes the formation of tight junction proteins ZO-1, claudin-1, and membrane transporters such as the Glut-1 and the ABC transporters [START_REF] Lee | SSeCKS regulates angiogenesis and tight junction formation in blood-brain barrier[END_REF]Liebner et al., 2011b;Paolinelli et al., 2011). The nuclear -catenin also induces the activity of the efflux transporters P-gP, Bcrp, Oat3 and Oatp1a4 through a mechanism that is dependent on the activation of endothelin-1 (Harati et al., 2013).

These studies showed that intricate interactions between the cells that compose the NVU are essential to induce the BBB phenotype in the endothelial cells. The development of the BBB starts during fetal life, however subsequent brain development during years in humans may change the BBB phenotype.

Brain Development

The development of the CNS in mammals occurs through a chain of events whose sequence is fairly conserved in a large number of species (Clancy et al., 2001(Clancy et al., , 2007;;Finlay and Darlington, 1995;de Graaf-Peters and Hadders-Algra, 2006). As a simple example, neuron proliferation precedes their migration and the development of axons and dendrites (de Graaf-Peters and Hadders-Algra, 2006).

This highly preserved sequence of events during CNS development allows the extrapolation of data between species, which is particularly important when animal models are used to study brain development (Clancy et al., 2007). In the case of this work, where pharmacoresistance to AEDs in childhood is suspected to be related to brain development, the use of animal models is important, in particular rodents. Hopefully, the comparison of CNS development between species is easier to do in the younger brain because later development during adulthood is majorly influence by the interaction with the surrounding environment (Clancy et al., 2007).

When comparing brain development between rats and humans, it was observed that in rats it occurs to a great extent after birth, while in humans it is considered a more perinatal phenomenon, that is, between the last trimester of pregnancy and the first two years of life (Dobbing, 1971(Dobbing, , 1974;;Dobbing and Sands, 1973, 1979, 1981;Winick, 1975). These initial studies looked into gross markers of brain development, such as the rate of gain in brain weight: it was concluded that the rat brain at post-natal day 8 (P8) was similar to human newborns (Dobbing, 1971(Dobbing, , 1974;;Dobbing and Sands, 1973, 1979, 1981;Winick, 1975). However, the comparison of the EEG records, which is a more robust analysis of brain function, suggests that a human newborn in more close to a P12 rat (Romijn et al., 1991). Further studying brain function, GABA inversion (a phenomenon occurring brain development, as previously described in section 2.2) occurs during the last trimester of pregnancy and in the first week of life in rats (until P7) (Clancy et al., 2007;de Graaf-Peters and Hadders-Algra, 2006;Quilichini et al., 2006;Romijn et al., 1991).

The consumption of energy substrates during brain development clearly distinguishes the adult and the developing brain. The immature brain is more dependent on simple energy substrates, such as ketone bodies, while the adult brain relies more on glucose as an energy supply. The Local cerebral metabolic rate of glucose consumption (LCMRglu) was studied in humans by PET-scan using 18 F-FDG ( 18 Ffluorodeoxyglucose) and in rats by autoradiography using 14 C-deoxyglucose. Human newborns showed LCMRglu values of 70% of adults that starts increasing in the 2-3 months after birth and peaks at twice the adult values at the age of 4 years until 10 years-old. Glucose consumption then starts to decrease during childhood until it reaches adult levels by 16-18 years-old (Chugani, 1998;Kinnala et al., 1996). When looking at rats, glucose consumption rapidly increases at P17 until P21, the age at each rats are weaned, although never attaining the high levels as observed in children.

Glucose consumption starts then to decrease where it reaches adult levels by P35 (Kinnala et al., 1996;[START_REF] Nehlig | Cerebral energy metabolism, glucose transport and blood flow: changes with maturation and adaptation to hypoglycaemia[END_REF]Nehlig et al., 1988[START_REF] Nehlig | Postnatal changes in local cerebral blood flow measured by the quantitative autoradiographic [14C]iodoantipyrine technique in freely moving rats[END_REF].

The rate of passage of the amino acid L-methionine through the BBB was studied during brain development. The amino acid is highly consumed in the immature brain, which further declines: adult values are reached in human at the age of 11 years and in rats at the end of the 4 week (P28) [START_REF] O'tuama | L-methionine uptake by human cerebral cortex: maturation from infancy to old age[END_REF]. The authors concluded interestingly that this reduction was caused by the reduction of the neutral amino acid transporter in the BBB [START_REF] O'tuama | L-methionine uptake by human cerebral cortex: maturation from infancy to old age[END_REF]. Another studied in rats where the incorporation of 35 S-methionine in the brain of rats showed gender-related differences after P35, which were associated with puberty [START_REF] Haar | Changes in protein content and in vivo incorporation of (35S)methionine into protein of discrete brain areas of the rat over puberty: sexual differences[END_REF].

The development of the CNS is also dependent on hormones produced outside of the brain. Thyroid hormones are important for stopping brain cell proliferation and stimulating differentiation [START_REF] Porterfield | The role of thyroid hormones in prenatal and neonatal neurological development--current perspectives[END_REF]. Its critical activity period on brain cells was found to be between the last trimester of pregnancy and the second year of life in humans, which corresponded in the rat between birth and P18-P21 [START_REF] Porterfield | The role of thyroid hormones in prenatal and neonatal neurological development--current perspectives[END_REF] .

These studies show some consistency between the brain development events in rats and humans. The following table shows the comparison between brain development in humans and rats. This allows considering a P12 rat similar to a newborn human, P14 to the beginning of infancy, P21 as children and between P28 and P42 as adolescents.

Development of the Blood-Brain Barrier's Functions

The studies concerning the appearance of the BBB during fetal life showed that the endothelial cells start to proliferate early in fetal life. This allows the distribution of nutrients to the more profound cells present in the developing brain parenchyma.

During fetal life, the fetus is protected from the environment by the maternal placenta, which allows the exchanges of substances between fetal and maternal circulation. This has led to the belief that the BBB at this stage does not need to protect the brain and is therefore considered as immature and leaky due to less complex tight junctions (Saunders et al., 2012). However, studies with dyes injected either through blood or directly in central nervous systems show little diffusion between the blood and the brain [START_REF] Ek | Barriers in the developing brain and Neurotoxicology[END_REF]. Moreover, electrical resistance, which correlates with low diffusion of ions through the paracellular space, increases from E17 to E21 were it attained almost adult levels (Butt et al., 1990). Fenestrations between endothelial cells are detected at E11 in the rat embryo but they almost disappear by E19, while in fetal mouse brains pinocytic vesicles diminish from E10 towards E17 (Bauer et al., 1993;Stewart and Hayakawa, 1994). The lesser complexity of the BBB tight junctions during fetal life may reflect the low hydrostatic pressure of the fetal blood and more evidence (reviewed in [START_REF] Ek | Barriers in the developing brain and Neurotoxicology[END_REF]Saunders et al., 2012;[START_REF] Strazielle | Efflux transporters in blood-brain interfaces of the developing brain[END_REF])

supports the hypothesis that the fetal an early post-natal BBB is functional and that differences between developing and adult BBB reflect however specific needs of the developing brain. Ek and colleagues actually stated that "The word immature should thus probably be used cautiously in this context since it is very easily misunderstood as indicating that barrier mechanisms in the developing animal are less effective than in the adult, that is, they are implied to be a less sophisticated version of the adult system" [START_REF] Ek | Barriers in the developing brain and Neurotoxicology[END_REF].

One interesting aspect of the developing brain is that it displays a brain barrier that does not exist in the adult brain: the brain-CSF barrier (Saunders et al., 2012). This barrier seems to restrict the passage of high molecular weight substances such as horseradish peroxidase, although its permeability to other molecules is not known [START_REF] Ek | Barriers in the developing brain and Neurotoxicology[END_REF].

More importantly to the BBB is the developmental expression of transporters in the surface of the endothelial cells. Different patterns of efflux transporters in the BBB between the developing and the adult brain may be related to different brain permeability of drugs. In post-mortem fetal brain human tissue, immunolocalization of P-gP, BCRP and MRP1 were observed in microvessels. While BCRP and MRP1 expression remained constant, P-gP appeared to increase its immunoreactivity in brain microvessels [START_REF] Daood | ABC transporter (P-gp/ABCB1, MRP1/ABCC1, BCRP/ABCG2) expression in the developing human CNS[END_REF]. After 12 weeks of gestation, P-gP immunoreactivity in human fetal brain was diffuse in endothelial cells, but with further development it was localized in the apical membrane and co-localized with caveolin proteins [START_REF] Virgintino | Fetal blood-brain barrier P-glycoprotein contributes to brain protection during human development[END_REF]. More studies have been made in animals, for instance expression of P-gP and Bcrp was shown to increase in the post-natal brain microvessels (Harati et al., 2013;[START_REF] Matsuoka | Developmental expression of Pglycoprotein (multidrug resistance gene product) in the rat brain[END_REF]. Immunoreactivity to P-gP is already observed in rat microvessels as early as embryonic day 9 (E9) that increased with brain development (Ek et al., 2010). Quantitative analysis of P-gP, Bcrp, Glut3 and Mct1 in the microvessels of developing and adult monkey brains showed an increase in Bcrp protein levels but a decrease in Glut3 and Mct1 transporters [START_REF] Ito | Quantitative membrane protein expression at the blood-brain barrier of adult and younger cynomolgus monkeys[END_REF]. Glut1 expression in fetal mouse brain was also shown to correlate with BBB tightness [START_REF] Bauer | Ontogenic expression of the erythroid-type glucose transporter (Glut 1) in the telencephalon of the mouse: correlation to the tightening of the blood-brain barrier[END_REF] and high levels of the Mct1 transporter were observed in the early post-natal microvessels of the rat brain (Baud et al., 2003). As previously said, brain uptake of methionine decreases with age, apparently caused by a developmental reduction of the BBB amino acid transporters [START_REF] O'tuama | L-methionine uptake by human cerebral cortex: maturation from infancy to old age[END_REF]. Older reports had already found a decrease in other neutral amino acids between P5 and P19 rats (Lefauconnier and Trouvé, 1983). GABA brain permeability was higher in the neonatal rat brain when compared with adults, and was related to higher levels of the GABA transporter in the BBB (Al-Sarraf, 2002).

Methods for the study of drug transport through the Blood-Brain Barrier

The understanding of the transport of xenobiotics through the BBB is currently a major concern in research. Especially regarding intentionally administered drugs, it is of most importance not only to understand how drugs cross the BBB but also how does this structure eliminate drugs in order to protect the brain parenchyma.

The study of the impact of the BBB on brain disposition of drugs in humans is a complex issue due to the difficulty of measuring the extent of brain penetration of a given molecule and to ethical concerns.

Many factors influence the brain disposition of intentionally administered drugs: cerebral blood flow, protein binding, plasma protein content, integrity of the BBB, metabolism or efflux in the BBB, and metabolism or efflux in the brain parenchyma cells (de [START_REF] De Lange | Considerations in the use of cerebrospinal fluid pharmacokinetics to predict brain target concentrations in the clinical setting: implications of the barriers between blood and brain[END_REF]. Any pathological setting that also impact one of these factors may therefore change drug disposition. Concentrations in cerebrospinal fluid (CSF) have been used as a surrogate of brain parenchyma concentrations because it is the only biological fluid that is in contact with brain cells and can be sampled. However, this fluid does not fully represent concentrations in brain parenchyma, especially in localized regions such as epileptic foci or tumors when CSF is collected through a lumbar puncture (de Lange and [START_REF] De Lange | Considerations in the use of cerebrospinal fluid pharmacokinetics to predict brain target concentrations in the clinical setting: implications of the barriers between blood and brain[END_REF]. Therefore, several animal models have been developed, both in-vivo and in-vitro, to study the transport of drugs through the BBB.

In vivo Models

In vivo models allow the maintenance of brain and BBB physiology; therefore the results obtained are closer to a real clinical setting. Nonetheless, the utilization of animals for this type of studies must be made cautiously because species differences in physiology can complicate the data transposition from animals to humans.

Brain Microdialysis

Microdialysis is a technique that allows the quantification of substances directly in the brain tissue. It consists of a very small catheter that is placed in the brain region where concentrations are to be measured, whose surface is made up of a semipermeable membrane that allows diffusion of molecules between the inside of the catheter and the surrounding interstitial fluid. Inside the catheter, a continuous flow of a perfusion fluid allows the recovery of the dialysate in a vial connected to the catheter [START_REF] Shannon | Cerebral microdialysis in clinical studies of drugs: pharmacokinetic applications[END_REF]. This allows the measurement of the unbound fraction of the drug in the brain.

Microdialysis has been also used as a technique in clinical studies: selected patients who were submitted to craniotomy for neurosurgical procedures had a catheter implanted [START_REF] Müller | Microdialysis in clinical drug delivery studies[END_REF].

Antiepileptic drugs have been investigated in these patients: in one group of patients submitted to epilepsy surgery, concentrations of AEDS were compared between microdialysate, serum, CSF and in the removed brain tissue, with little correlation between all these samples [START_REF] Rambeck | Comparison of brain extracellular fluid, brain tissue, cerebrospinal fluid, and serum concentrations of antiepileptic drugs measured intraoperatively in patients with intractable epilepsy[END_REF].

Nonetheless, the lack of controls in this study (either nonepileptic tissue or patients without epilepsy)

did not allow making assumptions regarding the possible resistance phenotype in the BBB.

Microdialysis in two patients with PHT showed however good correlations between interstitial fluid concentrations and unbound serum concentrations [START_REF] Scheyer | Phenytoin concentrations in the human brain: an in vivo microdialysis study[END_REF].

On the other hand, studies in animals can provide more information because controls can be used and experiments with crossover designs can be used. In the case of mice, transgenic animals can be used.

For instance, P-gP activity was analyzed, using rhodamine 123 as P-gP substrate in P-gP knock-out mice (mdr1a (-/-)) showing the impact of P-gP in brain distribution of xenobiotics [START_REF] Boschi | Microdialysis in mice for drug delivery research[END_REF]. Microdialysis studies in rabbits allowed the identification of different efflux mechanisms of VPA in the brain cells and the BBB [START_REF] Scism | Probenecid-inhibitable efflux transport of valproic acid in the brain parenchymal cells of rabbits: a microdialysis study[END_REF]. Furthermore, PB, LTG and FLB were identified as P-gP substrates in rats by using microdialysis [START_REF] Potschka | P-Glycoprotein-mediated efflux of phenobarbital, lamotrigine, and felbamate at the blood-brain barrier: evidence from microdialysis experiments in rats[END_REF].

Knock-out animals

Transgenic mice have been used to study brain penetration of AEDs. This technique allows understanding the full role of a transporter in brain disposition of a drug. The development of mice knock-out for P-gP (Mdr1a(-/-), Mdr1b(-/-)) and BCRP (Abcg2(-/-)) allowed the identification of AEDs that are substrates of these transporters (Nakanishi et al., 2013;[START_REF] Sills | Pglycoprotein-mediated efflux of antiepileptic drugs: preliminary studies in mdr1a knockout mice[END_REF]. Rats knockout for MRP2 allowed the identification of PHT as a substrate of MRP2 (Potschka et al., 2003). PHT was also shown to be substrate of P-gP in mice knock-out for mdr1a and mdr1b [START_REF] Sills | Pglycoprotein-mediated efflux of antiepileptic drugs: preliminary studies in mdr1a knockout mice[END_REF].

Positron-Emission Tomography

Positron-Emission Tomography (PET) is an image technique that allows the analysis of the distribution of a drug in the whole body. It is based on the utilization of radiolabeled molecules whose radioactive atoms disintegrate by β + decay. The emission of a positron (the β + particle) is detected in tissues after this particle annihilates an electron (the β -particle) in the vicinity of the molecule, which therefore releases γ radiation that is detected outside the body. Wherever the radiolabeled molecule accumulates in the body, higher emission of γ rays will be detected and thus can be quantified.

Only two radiolabeled AEDs have been studied through PET: [ 11 C]mephobarbital in rodents and [ 11 C]valproic acid in non-human primates. [ 11 C]mephobarbital was studied as a potential P-gP substrate but no evidence was reported as it being a P-gP substrate [START_REF] Mairinger | The antiepileptic drug mephobarbital is not transported by P-glycoprotein or multidrug resistance protein 1 at the blood-brain barrier: a positron emission tomography study[END_REF]. The distribution in the whole body of [ 11 C]valproic acid was analyzed, reporting accumulation in liver, spleen, heart and brain tissue [START_REF] Kim | Whole-body pharmacokinetics of HDAC inhibitor drugs, butyric acid, valproic acid and 4-phenylbutyric acid measured with carbon-11 labeled analogs by PET[END_REF].

Parallel to mephobarbital study discussed above, more studies have used PET to measure P-gP activity in the brain. [ 11 C]N-desmethylloperamide was used as substrate of P-gP for testing the impact of AEDs in P-gP function: LEV, TPM, and PHT were demonstrated as inducers of P-gP at therapeutic concentrations and not impacting P-gP at higher than therapeutic concentrations. VPA appeared not having any impact on P-gP (Moerman et al., 2011). In another study using R-[ 11 C]verapamil in healthy and pharmacoresistant patients with temporal lobe epilepsy, although no statistical difference was observed, there was a trend for P-gP activity to increase in epilepsy patients that could be responsible for resistance to AEDs [START_REF] Langer | Pharmacoresistance in epilepsy: a pilot PET study with the P-glycoprotein substrate R-[(11)C]verapamil[END_REF]. Seizures did not change the P-gP mediated efflux of

[ 11 C
]flumazenil in a kainate-induced model of epilepsy [START_REF] Syvänen | Altered GABAA receptor density and unaltered blood-brain barrier transport in a kainate model of epilepsy: an in vivo study using 11C-flumazenil and PET[END_REF].

In vitro Models

The construction of in vitro BBB models allows the recreation in the laboratory of the environment surrounding the cells that composes the BBB. These models are frequently constructed with cells that resemble brain endothelial cells with laboratory material (Transwell® inserts) that allows the polarization of these endothelial cells. In many in-vitro BBB models the creation of two compartments, apical and basolateral that mimic blood circulation and brain parenchyma, respectively, allows the study of the passage of substances between blood and brain.

BBB models based on brain endothelial cells

The models that are the closest to the real BBB environment are produced by using brain endothelial cells. These cells are frequently primary cultures of endothelial cells or low passage cultures obtained from fresh tissue. Endothelial cells from different species have been used: bovine, porcine, rodent, and more importantly, human cells. Primary cell cultures of brain endothelial cells are expected to maintain their in-vivo physiology, with the expression of tight junctions, metabolic pathways and influx and efflux transporters (Deli et al., 2005).

In most models of the BBB, brain endothelial cells are co-cultured with other cells in order to establish the physiological interaction observed in the brain (Harati et al., 2013;[START_REF] Josserand | Evaluation of drug penetration into the brain: a double study by in vivo imaging with positron emission tomography and using an in vitro model of the human blood-brain barrier[END_REF][START_REF] Lacombe | In vitro primary human and animal cell-based blood-brain barrier models as a screening tool in drug discovery[END_REF][START_REF] Mabondzo | Validation of in vitro cell-based human blood-brain barrier model using clinical positron emission tomography radioligands to predict in vivo human brain penetration[END_REF][START_REF] Megard | A co-culture-based model of human blood-brain barrier: application to active transport of indinavir and in vivo-in vitro correlation[END_REF]. Endothelial cells are therefore co-cultured with glial cells and/or pericytes. In some models these cells were replaced by conditioned medium: media from other brain cells cultures, such as astrocytes, is used as the basolateral compartment mimicking brain parenchyma (Cardoso et al., 2010a;Deli et al., 2005).

Human cells used in the establishment of BBB models are obtained from patients subjected to neurosurgical procedures, such as in the case of epilepsy surgery. Post-mortem samples have also been used to create these models. Although these cells would be expected to provide the best models of human BBB, the fact that they are obtained from pathological or post-mortem tissues may add bias to the obtained results. Moreover, even if these models could bring more information about the role of the BBB in these pathologies, such as data on brain distribution of antiepileptic or anticancer drugs, the lack of healthy controls (unobtainable due to ethical concerns) always hinders the generalization of the information.

BBB models were constructed with endothelial cells from operated epilepsy patients. These were frequently obtained from temporal lobe epilepsy patients [START_REF] Fiala | TNF-alpha opens a paracellular route for HIV-1 invasion across the blood-brain barrier[END_REF][START_REF] Jong | Traversal of Candida albicans across human blood-brain barrier in vitro[END_REF][START_REF] Mackic | Cereport (RMP-7) increases the permeability of human brain microvascular endothelial cell monolayers[END_REF][START_REF] Muruganandam | Development of immortalized human cerebromicrovascular endothelial cell line as an in vitro model of the human blood-brain barrier[END_REF][START_REF] Stins | Bacterial invasion and transcytosis in transfected human brain microvascular endothelial cells[END_REF]. In one study, cells were transfected with immortalizing genes in order to produce cell lines that could be used for transport studies through the BBB [START_REF] Muruganandam | Development of immortalized human cerebromicrovascular endothelial cell line as an in vitro model of the human blood-brain barrier[END_REF]. This model showed the presence of common endothelial markers such as γ-glutamyl transpeptidase or alkaline phosphatase, with reduced sucrose permeability (a marker of the integrity of the cell monolayer). Interestingly, in only one study endothelial cells from epileptic tissue were used to study the transport of AEDs [START_REF] Cucullo | Development of a humanized in vitro blood-brain barrier model to screen for brain penetration of antiepileptic drugs[END_REF].This in vitro BBB model was constructed with endothelial cells and glial cells from epileptic tissue. Transport of PHT and diazepam through this model was compared with transport through commercial endothelial cells and astrocytes cultured with the same technique. PHT passage from the apical to the basolateral compartment was significantly reduced in cells from epileptic patients. Moreover, inhibition of P-gP in the model built from epileptic tissue cells partly increased the passage of PHT [START_REF] Cucullo | Development of a humanized in vitro blood-brain barrier model to screen for brain penetration of antiepileptic drugs[END_REF].

The passage of drugs through the developing BBB has been the objective of only one study [START_REF] Takata | In vitro blood-brain barrier models using brain capillary endothelial cells isolated from neonatal and adult rats retain age-related barrier properties[END_REF] where in vitro models of the BBB were constructed. In this work brain endothelial cells from immature (post-natal day 14) and adult rats (post-natal day 56) were used. VPA passage was analyzed, comparing the brain-plasma permeability between in vivo and in vitro. Results showed a higher permeability of VPA in immature rats when compared to adults, both in vivo and in vitro.

BBB models based on other cell types

The construction of in-vitro BBB models has also been made with brain endothelial transformed cell lines. These cell lines are derived from primary cultures of brain endothelial cells that were transfected with viral genes. These genes allow the cell line to continuously multiply, which allowed the establishment of homogenous cells that display most of the characteristics of brain endothelial cells.

Cell lines from human origin (hCMEC/D3) and from other animals (rat GPNT and RBE4, or the mouse b.END.3) have been developed (Roux and Couraud, 2005;[START_REF] Roux | Regulation of gamma-glutamyl transpeptidase and alkaline phosphatase activities in immortalized rat brain microvessel endothelial cells[END_REF]. These cell lines however do not present all the characteristics of the BBB, particularly the reduced paracellular transport [START_REF] Omidi | Evaluation of the immortalised mouse brain capillary endothelial cell line, b.End3, as an in vitro blood-brain barrier model for drug uptake and transport studies[END_REF]. These cells form leaky monolayers, commonly measured by a reduced trans-endothelial electrical resistance (TEER) which reduces their convenience for transport studies (Deli et al., 2005;Roux and Couraud, 2005).

These transformed endothelial cells have been used for studies of the impact of AEDs in the efflux activity of selected ABC transporters. The impact of CBZ, LEV, PB, PHT, TPM and TGB on the expression of P-gP and MRPs was analyzed in the rat GPNT and RBE4 cell lines. The authors showed that CBZ, PB and PHT were more effective than the other AEDs in inducing the studied ABC transporters [START_REF] Lombardo | Induction of nuclear receptors and drug resistance in the brain microvascular endothelial cells treated with antiepileptic drugs[END_REF]. This induction was dependent on the activation of Pregnane X Receptor (PXR) and Constitutive Androstane Receptor (CAR) nuclear receptors and the effects were also dependent on the used cell line. A study to compare the effects of CBZ, LEV, PB, PHT, TPM and VPA on the efflux activity of P-gP was performed in transformed rat and human cells lines. The results were also compared with experiments primary cultures of rodent and porcine brain endothelial cells. Although the response varied between cell lines, CBZ apparently induced P-gP and VPA inhibited P-gP [START_REF] Liu | Impaired expression and function of breast cancer resistance protein (Bcrp) in brain cortex of streptozocin-induced diabetic rats[END_REF]. Nonetheless, the authors considered that little the AEDs have little impact on P-gP efflux activity that might be clinically relevant.

Objectives

As reviewed in the introduction, the differentiation of the BBB during brain development may have an important impact on the distribution of drugs to the brain. The differences in the expression of ABC and SLC transporters that may occur during brain development in the endothelial cells of the BBB could change the passage of the AEDs from blood to the brain parenchyma. Children with Dravet Syndrome are chronically exposed to pharmacotherapy in order to control seizures. As the expression of the major efflux transporters that occur in the BBB are influenced by xenobiotics such as drugs, when the BBB is exposed to chronic treatment with AEDs during development, changes in its pattern of ABC and SLC efflux transporters could be induced.

The objectives of this work are therefore:

1 -To review the published in vitro BBB models constructed with primary cultures of brain endothelial cells in order to verify if these models had taken into account age-related differences in the Concerning objective 3, in parallel to the AEDs used in the treatment of Dravet syndrome, carbamazepine was also studied as an example of AEDs used in the treatment of focal seizures.

Methods

Methods of literature review: "In vitro models of the immature blood brain barrier for optimizing drug disposition"

We searched for the published in vitro BBB models in the MEDLINE database with the key-words "blood-brain barrier" and "model". The output list was analyzed and cross-checked with a published review about the in vitro BBB models in order to select studies that were not found with primary search in the MEDLINE database. Only the publications related to the models constructed with primary cultures of brain endothelial cells were analyzed to verify if brain development was taken into account.

Methods for the study of the BBB ontogeny: "Ontogeny of ABC and SLC transporters in the microvessels of developing rat brain"

For this objective, rats of three classes of age were used: post-natal day 14 (P14) rats that are considered as comparable to infants (between 1 month and 2 year-old humans), post-natal day 21 (P21) rats considered as comparable to children (between 2 year-old and adolescence) and post-natal day 56 (P56) rats that we considered as adults. These age classes allowed us to cover the corresponding age-range of Dravet Syndrome patients: indeed seizures in Dravet syndrome frequently start in 6 month-old infants and continue through childhood until adulthood.

To assess the changes in the ABC and SLC genes expression in the developing BBB, semiquantitative Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) was performed to measure mRNA levels in the brain microvessels. This is a semi-quantitative method for measuring changes in mRNA levels, where the target gene is compared to an internal calibrator, the housekeeping gene, The study of the efflux activity of the transporter genes was performed for the two major efflux transporters in the BBB, P-glycoprotein (P-gP) and Breast Cancer Resistance Protein (BCRP). Efflux activity was calculated using the brain/blood partition coefficients of a substrate for each transporter in treated developing rats at plasma steady-state concentrations using osmotic minipumps. We selected digoxin (DGX) as a substrate for P-gP and prazosin (PRZ) as BCRP substrate.

Methods for the study of the impact of AEDs treatment in the developing BBB:"

Impact of the antiepileptic drugs used in the Dravet syndrome on the Blood-Brain Barrier in the immature rat brain"

The core pharmacotherapy of Dravet Syndrome consists in the triad of valproic acid (VPA), clobazam (CLB) and stiripentol (STP). These drugs were administered through osmotic minipumps (Alzet® minipumps) that allow the continuous infusion of the drugs and measurement of plasma concentrations at steady-state, that is, stable concentrations in plasma were obtained. This simplifies the calculation of the partition coefficients of each drug: plasma steady-state concentrations are in equilibrium with brain concentrations and brain-blood partition coefficients are conveniently calculated as the ratio of the concentrations in brain and plasma. Moreover, this method greatly reduces the manipulation of animals, reduced to the implantation of the minipumps, increasing animal well-being.

Because chronic treatment in developing animals with these drugs administered through osmotic minipumps has not been published yet in scientific literature, we performed a dose-plasmaconcentration study where we searched for the dose administered that originated the plasma concentrations considered as therapeutic in humans. Our goals in this objective were therefore to obtain plasma concentrations within the following intervals: VPA -[50, 100] mg/L; STP - [4,22] mg/L; CLB - [30;300] μg/L and its active metabolite N-desmethylclobazam nCLB -[0,3;3] mg/L.

Chronic treatment with the AEDs was limited to five days because this duration of treatment was considered as sufficient to obtain either induced or inhibited gene expression and not to long for each growing up animal to exclusively remain in its own age class. In the presented work we hypothesized that BBB maturation has an impact on the AEDs brain disposition. We therefore analyzed the passage of the AEDs used in Dravet Syndrome using as an end-point the brain-blood partition coefficient of each AED. In order to include the possible induction or inhibition mechanism by each AED during a chronic treatment in the developing BBB, to compare to the chronic treatment administered to humans, these end-points were calculated at the end of the five day treatment and compared between P14, P21 and P56 rats.

Gene expression was calculated as 2 -ΔΔCt , where ΔΔCt refers to difference between the threshold cycle of the target gene and Cyclophylin A (Ppia) as the control housekeeping gene in treated and control animals (ΔΔCt = ΔCt treated -ΔCt control , ΔCt= Ct target -Ct Ppia ).

The study of the efflux activity of the transporter genes was made for the two major efflux transporters in the BBB, P-glycoprotein (P-gP) and Breast Cancer Resistance Protein (BCRP). Efflux activity was measured indirectly by calculating the brain/blood partition coefficients of a substrate of each transporter in developing rats at plasma steady-state concentrations using osmotic minipumps. We selected digoxin (DGX) as a substrate for P-gP and prazosin (PRZ) for BCRP, which were infused together at the end of the five-day treatment of each AED, to calculate the end-point brain/blood partition coefficient of each substrate.

Impact of carbamazepine on the pattern of ABC and SLC transporters of the developing BBB

Carbamazepine (CBZ) is an AED frequently prescribed for patients with focal seizures is avoided in the treatment of Dravet syndrome because it can exacerbate seizures in these patients. CBZ was studied alongside the AEDs used to treat Dravet syndrome with the same methods as for VPA, STP and CLB. CBZ was administered through osmotic minipumps (Alzet® minipumps) that allow the continuous infusion of the drugs and measurement of plasma concentrations at steady-state, which allow the calculation of brain-blood partition coefficients are conveniently calculated as the ratio of the concentrations in brain and plasma. Chronic treatment in developing animals with this drug administered through osmotic minipumps has not been also published scientific literature, and therefore a dose-plasma-concentration study was also done for CBZ. For this AED, the therapeutic plasma concentration interval in humans is CBZ - [4,12] mg/L. In parallel with the studied AEDs, treatment was also limited to five days.

Gene expression was calculated as 2 -ΔΔCt , where ΔΔCt refers to difference between the threshold cycle of the target gene and Cyclophylin A (Ppia) as the control housekeeping gene in treated and control animals (ΔΔCt = ΔCt treated -ΔCt control , ΔCt= Ct target -Ct Ppia ).

The study of the efflux activity of the transporter genes after CBZ treatment was made once more for the two major efflux transporters in the BBB, P-glycoprotein (P-gP) and Breast Cancer Resistance Protein (BCRP). Efflux activity was measured indirectly by calculating the brain/blood partition coefficients of digoxin (DGX) as a substrate for P-gP and prazosin (PRZ) for BCRP, which were infused together at the end of the five-day treatment with CBZ, to calculate the end-point brain/blood partition coefficient of each substrate.

Interaction of VPA with the efflux transporters P-gP and BCRP in an in vitro model of the developing BBB constructed with primary cultures of rat brain endothelial cells

The influence of VPA on P-gP and BCRP efflux activity was further analyzed in an in vitro model of immature BBB. This model was constructed with primary cultures of rat brain endothelial cells from animals at the post-natal day 21. Brain microvessel endothelial cells were isolated from immature and adult rat brains after euthanasia under pentobarbital anesthesia and brains collected in cold HBSS -1%

PSN buffer (Hanks Balanced Saline Solution supplemented with penicillin, streptomycin and neomycine). Cell isolation was started by clearing the cortex from cerebellum, meninges, surface vessels, and brainstem. After homogenization, the tissue was digested with collagenase/dispase (1mg/mL) (Roche Diagnostics) in HBSS -1% PSN solution, supplemented with 10UI/μL DNase and 1μg/mL TLCK (N-tosyllysine chloromethyl ketone, Sigma Aldrich), for 1h at 37ºC. The digested tissue was pelleted and ressuspended in 20% bovine serum albumin in HBSS -1% and centrifuged at 2900rpm for 30min at 4ºC. The pelleted microvessels were separated from the overlaying myelin further digested for 1h with collagenase/dispase (1 mg/mL) in HBSS -1% PSB solution, supplemented with 10UI/μL DNase and 1μg/mL TLCK at 37ºC. Endothelial cells were seeded in T75 flasks previously coated with collagen IV (Sigma Aldrich) and fibronectin with EBM-2® medium (Lonza) supplemented with the EGM-2 MV ® SingleQuots (Lonza) and 3 μg/mL puromycine (Sigma Aldrich). Cells were kept at 37ºC at 5% CO2, the medium changed every other day first for EBM medium with 2 μg/mL puromycine and subsequently for EBM medium, and trypsinized when 80% confluence was reached. Cells were frozen in liquid nitrogen with 10% DMSO in fetal bovine serum until further use.

Glial cells were obtained from P2 rats similarly as for endothelial cell isolation, but the myelin supernatant was collected and diluted in HBSS-1% PSN buffer and centrifuged at 1500rpm. Pelleted glial cells were ressuspended in AST medium, composed of 1:1 mix of MEM-α and F-12 nutrient mix (both from Gibco) supplemented with 5% fetal bovine serum (Lonza), 1% PSN (Sigma Aldrich), 1%

human serum (Sigma Aldrich), and 0.4% fibroblast growth factor (Lonza); and seeded in T75 flasks coated with 0.1% poly-L-lysine (Sigma Aldrich) aqueous solution. Cells were kept in an incubator at 37ºC and 5% CO2, the medium changed every other day with AST medium, and trypsinized at 80% confluence. Cells were stored in liquid nitrogen with 10% DMSO in fetal bovine serum.

Primary endothelial and glial cells co-culture in Transwell® inserts started at day -1 seeding glial cells in 12-well plates in AST medium (20.000 cell/well). At day 0, polycarbonate Transwell® inserts (1.12 cm 2 , 0.4 μm pore, Corning) were coated with 0.1mg/mL collagen IV and 10μg/mL fibronectin (Sigma Aldrich), seeded with endothelial cells (80.000 cell/insert) in END medium, composed of EBM-2 medium supplemented with EGM-2 MV SingleQuots (without VEGF), and inserted in the glial cells' seeded wells, thus creating an apical (A) compartment mimicking blood current and a basolateral (B) compartment mimicking brain parenchyma. Media were changed every other day, removing all apical medium and half of the basolateral medium, and cell monolayer integrity was followed by transendothelial electrical resistance (TEER). When TEER levels reached 150 Ω/cm 2 , endothelial monolayers were validated by measuring 14 C-sucrose and 3 H-vinblastine permeability. Apparent permeability of each substrate was calculated according the following equation:

Endothelial cells monolayer in

where P app is apparent permeability, dQ/dt is the quantity of AED detected in the receiver compartment per time point, A is the area of the insert and C 0 is the concentration of the AED at the t = 0 min. Efflux ratios (ER) were calculated with the following equation:

Results

Results of the exhaustive review of the literature on the topic of in-vitro BBB models

The implication of the BBB in several neurological pathologies has led researchers to develop in vitro models of the BBB. These models have been useful not only to study the pathophysiology of brain diseases but also to study the transport of substances between the blood and the brain. Particularly regarding drug development, in vitro models of the BBB have been regarded as a much useful tool for screening of potential drugs for brain diseases.

The basic architecture of these models consists of a monolayer of cells that is seeded in a semipermeable membrane allowing the creation of two compartments: the basolateral compartment which mimics the brain parenchyma and the apical compartment that resembles blood circulation. The usefulness of these models is therefore dependent on the capability of creating a monolayer of cells with restricted paracellular transport and establishing a cell phenotype that has the same properties of brain endothelial cells.

Many types of cells have been used for the construction of BBB models. The commonest are tumor derived cell lines, transformed brain endothelial cell lines and primary cultures of brain endothelial cells. From this group, primary brain endothelial cells are supposed to be the most reliable since they originate models presenting more restrictive paracellular transport and a pattern of efflux transporters closer to the in vivo phenotype. These models are frequently constructed with cells from a bovine, porcine, or rodent origin; nonetheless cells from human tissue, obtained from autopsy specimens or brain tissue of patients submitted to neurosurgical procedures, have been used in BBB models.

The assumption that primary cultures of brain endothelial cells can replicate more closely the actual phenotype of the in vivo BBB allows the hypothesis that the potential age related differences in the BBB might be reflected on the in vitro models. One could therefore develop a model to study drug transport through the developing BBB if the endothelial cells were obtained from animals at different stages of brain development.

We performed a review of the published in vitro BBB models, produced fom primary cultures of brains endothelial cells, in order to identify the studies where age related differences in BBB permeability were taken in account. This was made by searching the MEDLINE database for the keywords "blood-brain barrier" and "model", and the selected studies were cross-checked with current review on BBB models (Deli et al., 2005).

The analysis of the resulting list of models showed that the information concerning the age of the endothelial cells was absent or poorly defined, imprecise terms such as "calf" or "less than 12 months"

were used. Only one study until present intentionally compared two models of in vitro BBB from immature and adult brains.

The pattern of efflux and influx transporters may modulate the transport of drugs through the BBB. If this pattern changes with age, differences in drugs brain permeability may be more difficult to identify when the in vitro BBB models do not clearly define the age of the endothelial cells. Age-related differences have already been identified for the SLC transporters, such as Glut-

-Introduction

The Blood-Brain Barrier (BBB) is an essential element in animals with a well-developed Central

Nervous System (CNS). It is responsible for creating a proper environment for normal brain function:

it reduces the passage of potentially toxic molecules from blood to the brain and keeps nutrients' concentrations in homeostatic levels. In higher vertebrates such as mammals it consists primarily of a continuous layer of endothelial cells while in animals with low cerebral vascularisation (e.g. insects) it consists of glial cells 1 . The more primitive barriers are only composed of glial cells and it is possible to observe in earliest embryonic stages of higher vertebrates a barrier formed by neuroependymal cells.

The appearance of a BBB was probably necessary to control the ionic environment surrounding synapses when more complex functions were developed 1 . In the mature mammal brain, an unbroken capillary endothelial cells monolayer form an impermeable barrier that blocks direct diffusion of blood components to the brain parenchyma; this is achieved through the establishment of tight junctions between endothelial cells. This endothelial barrier is surrounded by other types of cells, astrocytes, neurons and pericytes, and the basement membrane creating the NeuroVascular Unit (NVU) 2 .

The development of a BBB in mammals starts before birth when endothelial cells increase the number of tight junctions, gradually creating an impermeable monolayer 3 . However, the development of BBB functions is still unclear: little information is known on the effects of maturation of neurons and astrocytes on the development of the BBB functions. Furthermore, when comparing data from different species it must be taken into account that at least in mammals the time course of brain maturation varies accordingly to the different species 4 .

The study of the BBB remains a top research subject because different CNS diseases appear to be a consequence of BBB deregulation or the diseases have an impact in the normal BBB phenotype.

Stroke and intracerebral hemorrhage are a direct consequence of BBB dysfunction 5 . Epilepsy has been linked to the BBB either as pathological step during status epilepticus or as having an impact in antiepileptic drugs delivery to the brain 6-9 . Expansion of gliomas or multiple sclerosis has been linked

to the BBB 10,11 . Moreover, the impact of BBB dysfunction in neonatal diseases such as perinatal stroke or infectious diseases still remains not fully understood 12,13 .

In the present review, we will clarify the influence of brain maturation on the BBB. Published BBB models will be reviewed in order to elucidate if brain maturation was taken into account in those studies.

2 -Cell types of BBB.

Endothelial Cells

The brain endothelial cells (BECs) that form the BBB have distinct characteristics from other endothelial cells: they present a higher number of tight junctions which obstruct the passage of solutes and water between cells; a higher concentration of mitochondria; they have a low level of pinocytic vesicles and display a specific pattern of transporter proteins, both solute carrier proteins from the SoLute Carrier family (SLC) and efflux pumps from the ATP binding cassette (ABC) family, on their surface 2 . These features reduce the passage of blood-carried substances either by reducing diffusion between cells, the paracellular route, and transport through cells, the transcellular route 14 . Tightjunctions structure in BECs are considered to be more complex than in other endothelial or epithelial cells 15 . They were first analyzed through freeze-fracture techniques that revealed the external fracture face (E-face) and protoplasmic fracture face (P-face) 1 . Tight-junctions are built on specific transmembrane proteins such as claudin, occludin and junctional adhesion molecules (JAMs) that link neighbor cells. These structures are then connected to the cellular actin cytoskeleton through cytoplasmatic proteins, the Zona occludens protein-1 (ZO-1), ZO-2 and ZO-3 16 .

These proteins belong to the Membrane-Associated Guanylate Kinase proteins (MAGUK) and may be subjected to regulation through serine kinases and protein C kinase 2,16 . Particularly, occludin subcellular localization is regulated by serine kinases-mediated phosphorylation, where its phosphorylation increases its presence in tight-junctions 1,17 . BECs are also connected through adhesion junctions who inhibit cell proliferation and also regulate paracellular permeability. Cadherin proteins are a family of transmembrane glycoproteins responsible for cell-cell adhesion that are dependent on Ca 2+ levels. Similarly to tight junctions' transmembrane proteins, cadherins are anchored to the intracellular actin cytoskeleton through other proteins, catenins 18 .

Pericytes

Pericytes are present in capillaries, arterioles and venules of many organs but are more frequently found in brain capillaries. They surround EC and are also covered by the basal lamina of the BBB.

Recent research has uncovered their function on brain capillaries: they seem to be responsible for controlling blood flow since they are susceptible to vasoactive molecules (e.g. catecholamines and nitric oxide) and have contractile properties 19,20 . Pericytes seem also important for brain angiogenesis as they actively interact with EC through the platelet derived growth factor receptor β (PDGFR β)

possibly to maintain a constant capillary diameter: PDGFβ was found to be a very important paracrine factor secreted by BECs for neovascularisation 20 .

Astrocytes

Further increasing the complexity of the NVU, astrocytic end-feet are in close contact with the endothelial monolayer and seem to have important actions on BBB function maintenance 21 . They secrete proteoglycans to form the NVU basement membrane but more importantly they secrete paracrine factors that modulate ECs barrier properties: when EC are co-cultured with astrocytes, increased levels of tight junction proteins ZO-1 and occludin are observed which increase the barrier tightness 2 . Examples of paracrine factors important on BBB homeostasis are tumor growth factor-β (TGF-β) or glial-derived neurotrophic factor (GDNF) 18 . Freeze-fracture techniques have also exposed some features of astrocytic endfeet surface: a high density of orthogonal arrays of intramembranous particles (OAPs) is present in areas of close contact with the basal lamina, which dramatically decreases when distance to the basal lamina increases 15 .

Neurons

Despite the consistent presence of neuron axons on the NVU, their precise role is not fully understood.

Capillaries and astrocytic endfeet are in direct contact with neuronal axons and, during brain development, neuroectodermal cells are responsible for the production of a VEGF gradient required for brain neovascularisation 2 . Furthermore, capillary blood flow may be controlled by neurons that innervate the NVU through the release of the vasoactive neurotransmitters such as acetylcholine and catecholamines and production of nitric oxide 22,23 .

-Experimental BBB models

Given its important functions, the study of the BBB has been directed towards its role in the selection of molecules to access the brain, its properties in metabolizing xenobiotics and the impact of BBB dysfunction on brain diseases 24 . While expressing specific transporters and efflux pumps at the surface of EC, the BBB protects brain parenchyma from potentially toxic xenobiotics but it also restricts the access of intentionally administered drugs to treat brain diseases. In order to study drug transport across the BBB and the effects of potentially toxic substances on the barrier integrity, in vitro models of the BBB have been developed to recreate the in vivo barrier environment. The common feature to all BBB models is the production of a tight monolayer of cells from endothelial origin.

BBB models are constructed using cells that have an endothelial phenotype. They are obtained by two procedures: isolation of endothelial cells from brain tissue or transformed endothelial cell lines.

3.1-Transformed endothelial cell lines.

Cell lines are produced through transformation of primary endothelial cell lines with immortalizing genes, e.g. the SV40 T-antigen 25 . The major drawbacks of its use in BBB models are the formation of leaker monolayers (higher permeability to solutes such as sucrose) and the lower expression of specific BBB markers (e.g. GLUT1 or P-gP) 25,26 , rendering the results difficult to correlate with in vivo. Some cell lines are also derived from non-cerebral endothelial cells 27 After obtaining the primary culture, endothelial cells are then cultured in plastic porous membranes, made from specific polymers (e.g. polycarbonate, polyester or polytetrafluoroethylene), which are usually collagen coated for proper cell proliferation. Transport studies are commonly accomplished in 0.4µm pore membranes. The BBB model is finally constructed placing this membrane in a culture well, in contact with a proper medium or co-cultured with other cell types (astrocytes or glioma cell lines, pericytes).

3.2-Isolation of endothelial cells from brain tissue

These models were only made possible after the first isolation and culture of endothelial cells 28,29 .

They are commonly isolated by first mechanically disrupting the brain tissue, followed by an enzymatic step and gradient centrifugations in order to separate the microvessel fragments 30 . A method consisting of only mechanical disruption and filtration has also been published 31 . Isolated EC should not be obtained because they do not form confluent monolayers 24 . The endothelial cell primary culture originating from the isolated microvessel fragments must be performed in collagen coated flasks for proper multiplication. Pericytes are normal constituents of the BBB and therefore are always collected during microvessel isolation. Their presence in the model endothelial monolayer causes its disruption and a leak model emerges, because endothelial cells surround pericytes with whom they do not form tight junctions 25 . They are efficiently eliminated from the primary endothelial culture by adding puromycine to the culture medium (pericytes lack P-gP efflux protein in their membranes and therefore are not able to reduce the toxic puromycine intracellular concentrations) 32 . However, when cultured in a separate monolayer, pericytes actually increase BBB tightness 33,34 .

Primary cells derived from animals (bovine, murine, porcine or rat endothelial cells) or humans (submitted to surgery or autopsy, but also fetal tissue) have a phenotype closer to an in vivo environment 2 provided that they are used with a low number of passages (no more than 8) 24,35 .

3.3-Criteria of validation of BBB

Reproducible BBB models should respect essentially four criteria: restrictive paracellular diffusion, physiological realistic cell phenotype, maintained transporter mechanisms, and high throughput (related to the endothelial cells' accessibility) 27 . The parameters that are most used to assess BBB paracellular diffusion are TEER and permeability studies 2 . A reliable BBB model should have a high TEER and low permeability to solutes that would only cross the BBB through a paracellular route.

TEER reflects the transport of ions through the endothelial barrier: a high TEER value correlates with less ion movements, the consequence of a high number of tight junctions which block the paracellular route. Standard accepted TEER values in vivo which were first observed in frog brain microvessels are around 1870Ω.cm 2 , although frog BBB lacks the astroglial envelope 36 . In rats, TEER increased from average 310Ω.cm 2 at E17 to a stable adult value of 1215 Ω.cm 2 at E21 37 .

Permeability measurements are more accurate than TEER to evaluate BBB tightness. A greater number of tight junctions in the BBB model endothelial monolayer increase the capacity of the model to discriminate between low and high permeability drug 38 . When a substance that only diffuses through the BBB by a paracellular route is used, the molecule permeability is inversely correlated with the BBB model tightness. Permeability measured in a BBB model is in fact the sum of three parameters: paracellular permeability, transcellular permeability and the aqueous diffusion (related with de diffusivity of a substance and the thickness of the aqueous boundary layer) 38 . The most common used substances are sucrose and sodium fluorescein and larger molecules such as albumin or inulin. For example, sucrose apparent permeability in the rat BBB after intravenous injection is about 0.03x10 6 cm.s -1 39 . The closer the in vitro permeability of the reference substance is to in vivo, the more discriminative the BBB model is.

3.4-BBB models from stem-cells

Recently, human pluripotent-induced stem-cells (hiPSC) have been used to develop BBB models 40,41 .

IMR90 fetal fibroblasts were transduced with lentivirus containing the OCT4, SOX2, NANOG, and LIN28, and were shown to present pluripotent phenotype similar to human embryonic stem-cells. One of the clones, hiPS-IMR90-4, was chosen to be differentiated into endothelial cells that when cocultured in Transwell® inserts with rat astrocytes displayed several features of an intact BBB.

Measured TEER values were in the range of 700-1450 Ω.cm 2 , cells expressed high levels of several ABC and SLC transporters genes, and permeability studies with both low permeability and high permeability molecules showed a good correlation with in-vivo data. Further development with the inclusion of retinoic acid and optimization of cell density resulted in a model that seemed a promising strategy for high-throughput testing of BBB permeability for new drugs targeting the CNS 42,43 .

-Methods

We searched for the published in vitro BBB models in the MEDLINE database with the key-words "blood-brain barrier" and "model". The output list was analyzed and cross-checked with a published review about the in vitro BBB models in order to select studies that were not found with primary search in the MEDLINE database 44 . Only the models constructed with primary cultures of brain endothelial cells were analyzed for the age of the animals used for the extraction of brain endothelial cells.

-Animal BBB models

Published BBB models (Table 1) were selected in order to evaluate if age related differences of cells that are used for the construction of these models were taken in account. The majority (45/84) of the published BBB models does not mention the age of the EC. Whenever EC age is recorded it was never considered as a factor influencing results. EC age is however more frequently mentioned when rat EC are used (16/20) probably because they originate from laboratory animals where development is controlled. In some models where EC age is stated it is not precisely defined, rather either with a wide range of ages (e.g. 1-3 months 45 , > 12months 46 ) or vague terms (e.g. calf for bovine EC 47 ); other authors select animals by their weight, from which their age can be inferred when good weight-age correlations are established 48-50 . Strikingly interesting is the fact that most of the AC used is from very young animals or even fetal AC (36/41). We can speculate that this is due to a higher production of paracrine factors in young AC where BBB is still being formed. It can be concluded that the published BBB models can be biased as they almost never consider that brain maturation may impact BBB functions. Takata et al. published a BBB model where differences between young and adult BBB was for the first time explored 51 . They compared two BBB models, one constructed with EC from P14 rats (considered as neonatal) and the other with EC from P86 cells (the adult cells). The authors reported low TEER values and high permeability to paracellular transport markers of the immature BBB model when compared with the adult model, concluding that the younger model was more permeable than the adult. These results were compared with in vivo data, where parallel results were observed, although no proper in vivo/in vitro correlation was made. Nonetheless, the reported TEER values of the immature BBB model were low: therefore this model may also be more permeable because it was no fully optimized.

-Evidence of blood brain maturation during childhood period

-Brain Growth

Central Nervous System ontology in mammals consists of a sequence of events highly conserved in a great number of species 52-55 . Generally, an initial period of clonal expansion is followed by functional differentiation and localization 53 . Considering neurons, for example, neuronal multiplication precedes axonal elongation and dendrites development 54 .

This sequence of events common to all mammals gives us the possibility to extrapolate data from one species to another 55 . Particularly during nervous system development the perinatal period is the most appropriate to establish a relationship between species, as further brain development in adults is more dependent on the interaction with the environment 55 .

Brain development is characterized by two major events of cell multiplication that are responsible for the increase in brain weight: neuronal and glial multiplication 56 . Clonal expansion of neurons during gestation in mammals is followed by the spread of glial cells that peaks around birth 57 . This glial "growth spurt" is the major responsible for the increase in brain weight 4 but its time position relatively to birth varies between species. When comparing rats with humans, this glial growth spurt is mainly achieved after birth, whereas in humans this phenomenon begins in the last trimester of pregnancy and continues in the two first years of life 4,56-59 . To further clarify this correlation between the growth spurts of these different species, studies analyzing the increase in brain weight during early maturation proposed rat post-natal day 8 (PN8) as equivalent to a newborn human 4,56-59 . Moreover, GABA activity inversion, an event that occurs in humans during the last trimester of pregnancy where GABA excitatory actions are inverted to the inhibitory activity seen in adults, is completed in rats during the first post-natal week (PN 2-7) 54,55,60,61 . However, when comparing the electroencephalographic record between newborn rats and humans, the appearance of the typical human newborn pattern known as "tracé alternant" occurs at PN 12-13 in rats 60 .

The correlation between glial growth spurts in different species is extremely important for the study of BBB development using animal models, as brain vasculature formation is strongly dependent on astrocytes. They secrete paracrine factors that regulate angiogenesis and BBB maturation 21 : some published BBB models have described the importance of endothelial cells and astrocyte cells coculture for the construction of reliable BBB models 34,62,63 . Others have evaluated the effect of astrocyte-conditioned medium (ACM) on endothelial cells cultures 64 . Particularly in rodents, as their glial-related growth-spurt is entirely post-natal, complete coverage of capillaries by astroglial end-feet is only reached at P21 23 .

-Paracrine Factors

Vascular Endothelial Growth Factor (VEGF) induces angiogenesis in the developmental brain by driving endothelial expansion to occur following a VEGF gradient 21 . VEGF was also shown to decrease BBB tightness, measured by the reduction of TEER and the increase in sucrose permeability; these events were correlated with a 32% decrease of occludin protein levels 34,65 . Other paracrine factors, such as Angiopoetin-1 and -2 (Ang-1 and Ang-2) are also involved in the angiogenic sprouting of endothelial cells 16 but have rather a tightening effect on endothelial monolayers 66,67 .

Though some paracrine factors have been identified regarding BBB maturation the exact mechanisms whereby the different type of cells communicate are still not fully understood. Wnt proteins appear to have a substantial role in the differentiation of the BBB 16 . Wnt proteins are a group of extracellular matrix glycoproteins that induces the phosphorylation of β-catenin through the canonical pathway mediated by Frizzled (Fzd) and Lipoprotein receptor-related protein cell surface receptors 68 . Stable βcatenin through phosporylation translocates into the nucleus where it induces gene expression. Wnt/βcatenin pathway was linked to BBB differentiation through the increase of claudin-3 expression 69 . Srcsuppressed C-kinase substrate (SSeCKS) is apparently a key pathway through which astrocytes regulate BBB maturation: c-Jun terminal kinases (JNK) phosphorylation, which are responsible for increasing VEGF concentrations and decreasing Ang-1, is inhibited by astrocyte SSeCKS 70 . Glial-cell derived neurotrophic factor (GDNF) was also shown to reduce BBB permeability, but only in the presence of high concentrations of cAMP, thus suggesting cAMP dependent pathways (BBB104).

Platelet Growth Derived Factor β (PDGF-β) and angiopoetins seem also to be excreted by endothelial cells in order to recruit pericytes to the neuro-vascular unit 16,20 .

-Transporter Proteins

The importance of astrocytes in BBB formation can be extended to the BBB functions: if BBB tightness is dependent on astrocytes, transporter mechanisms such as efflux pumps, solute transporters and metabolizing enzymes are possibly influenced by paracrine factors. The fact that glial expansion in humans continues through the two first years of life may result in different age-related patterns of transport mechanisms and metabolism, when compared with adults. Therefore, many BBB functions, such as ion regulation or nutrient uptake, may continue to maturate after birth 14 .

Wnt7a subfamily proteins were shown to induce glucose transporter 1 (GLUT-1) in mice embryos 71 .

Nonetheless, lactate and other ketone bodies (e.g. acetoacetate and β-hydroxybutyrate) appear to have a very important role as an energy substrate in the developing brain: monocarboxylate transporters 1 and 2 (MCT1 and MCT2) are more expressed during early brain development in mice, with a peak around PN15, until PN30 where they attain adult levels 72 . This higher expression compared to adults was observed not only on endothelial cells but also on pericytes and astrocyte end-feet 73 .

Early reports uncovered that immature brains have a greater capacity to uptake some aminoacids, such as alanine, serine, cysteine and threonine, than mature brains 74 .Co-culture of bovine endothelial cells with astrocytes showed the induction of the expression of the EAAT3 (Excitatory Amino-Acid

Transporter 3) one of the glutamate transporters 27 . When analyzing L-Methionine brain uptake between humans and rats, it was observed a continuous decrease in its passage (measured by PET) from birth to four weeks after birth in rats and until 10-11 years in humans 75 .

Nonetheless, comparisons between species must be made carefully. Differences in efflux transporters protein levels between rats and monkeys were observed: whereas cynomologous monkeys have higher levels of BCRP, in rat P-gP and MRP4 protein levels are higher 76 . Differences in ABC transporters expression between immature and adult brains were also identified: P-gP expression seemed to increase between the first days of life until childhood and then drop to neonate values in adulthood, while BCRP protein levels increased from the neonatal period until adulthood 76 .This data was however obtained from very few individuals and should be therefore further verified.

-Conclusion

Brain development must be considered as a process that affects all types of brain cells and not only neurons and their functions. As seen before, the formation of the BBB is intimately linked to the other brain cells through paracrine factors. The BBB most probably follows the development of neurons and astrocytes by changing its barrier function and pattern of transporter proteins in the surface of the EC.

Therefore, the construction of BBB models must ponder the possible effects of cells' age specially when they are used for drug transport studies. This will also help in the development of pharmacological therapies for pediatric populations, as these in vitro models will specifically try to recreate the environment of the BBB in the different pediatric ages and could be used to elucidate drug transport mechanisms in order to optimize drug disposition. The BBB has been associated with resistance to antiepileptics (AEDs). As the BBB present many transporters and the AEDs must cross the BBB to exert their action on neurons, researchers have focused on the dysfunction of the BBB as a cause of epilepsy, the identification of the AEDs who are substrate of efflux transporters, and the association of polymorphisms of efflux transporter genes with pharmacoresistant epilepsy. Considering that pharmacoresistance is a major concern in child epilepsy, and supposing that the developing BBB may present a pattern of transporters different from an adult brain, we explored in this article the ontogeny of selected ABC and SLC genes in the microvessels of P14, P21 and P56 rats. This was done by analyzing mRNA levels of the selected genes in the microvessels of the three classes of age by semi-quantitative RT-PCR. Genes were selected by their importance on the efflux of xenobiotics at the BBB, their selectivity for AEDs, or their implication on epilepsy. We studied also the efflux activity of the major ABC efflux transporters in the BBB, Pglycoprotein (P-gP) and Breast Cancer Resistance Protein (BCRP), in the three classes of age. This allows inputting other regulatory mechanisms that control gene expression, such as alternative splicing, mRNA stability, protein stability or direct allosteric inhibition and induction on P-gP and BCRP transporters.

mRNA levels were semi-quantified by RT-PCR in freshly isolated brain microvessels. Brains of P14, P21 and P56 rats were collected after euthanasia of animals under deep isoflurane anesthesia. After removing the meninges, large vessels and white matter, brain tissue was minced and digested with a solution of collagenase/dispase enzymes. In order to separate microvessels from neurons and glia, the digested tissues was centrifuged with a 20% albumin solution. mRNA levels quantification was preformed with conventional methods: extraction of total RNA by the TRizol® reagent followed by purification in RNA binding columns, cDNA synthesis from total RNA and semi-quantification by real-time PCR. Recorded threshold cycles of the target gene and the control housekeeping gene (cyclophylin A, Ppia) allow calculating the 2 -ΔCt values.

For analysis of P-gP and BCRP efflux activity, brain-plasma partition coefficients (K p, brain/plasma ) were determined for digoxin (DGX), a P-gP substrate, and prazosin (PRZ), a BCRP substrate. Both molecules were administrated concomitantly through a sub-cutaneously implanted osmotic minipump in rats belonging to each of the three classes of age. After reaching steady-state plasma concentration, rats were euthanized under deep isoflurane anesthesia, whole blood and brains were collected for dosage of DGX and PRZ by liquid chromatography coupled to mass spectroscopy (LC-MS).

Considering the expression of the ABC transporters genes, we observed that maturation of endothelial cells present in brain microvessels from rats P14 towards P56 resulted in an increase in the expression of Abcb1a and Abcg2 genes, and a decrease in Abcb1b gene, whose expression was clearly lower than Abcb1a. We did not detect Abcc2 mRNA in any class of age that was studied (Figure 1, see article).

When regarding SLC genes, differences were limited to the higher levels of gene Slc16a1 in P14 rats that steadily decreased towards adult levels (Figure 3, see article).

The effect of maturation in the P-gP and BCRP efflux activity was measured by its effect on the K p, brain/plasma of DGX, a P-gP substrate, and PRZ, a BCRP substrate. We observed an increase in P-gP efflux activity from P14 rats towards P56 rats, as measured by a decrease in the K p, brain/plasma of DGX.

No difference was observed concerning BCRP efflux activity.

The increase in P-gP efflux activity correlates with the increased expression of the Abcb1a gene. In contrast, BCRP efflux does not correlate with the Abcg2 gene expression, suggesting posttranscriptional modulation of the Abcg2 gene product. Slc16a1 gene is related to the supply of energy substrates, such as lactate and other monocarboxylates, in the developing brain. Moreover, Mct1, the product of the Slc16a1 gene has been shown to mediate the transport of VPA. As these genes have been associated with the efflux of AEDs (Table 1, see article), these maturational changes in the expression and function of the efflux transporters may therefore modulate the disposition of the AEDs in brain. These genes should therefore be accounted when studies concerning the role of the developing BBB in the pharmacoresistance in childhood epilepsies are performed. Furthermore, the impact of the AEDs in the developing BBB should also be analyzed, particularly the AEDs used to treat Dravet Syndrome because this is a good model of a childhood epilepsy where pharmacoresistance frequently appears.

Ontogeny of ABC and SLC transporters in the microvessels of developing rat brain

Introduction

During development in mammals, the brain goes through various stages from the formation of the neural tube to the adult stage. Brain development is characterized by sequential steps: an initial step of cell proliferation followed by cell migration and finally differentiation [1]. The Blood-Brain Barrier (BBB), the specialized interface between the vascular compartment and the brain parenchyma, develops in a similar fashion: angiogenesis first proceeds in a radial fashion, elicited by paracrine factors such as VEGF (Vascular Endothelial Growth Factor) released by neurons and astrocytes [2],

which is then followed by differentiation/maturation steps. In this last phase, endothelial cells acquire the most important BBB features: tight junctions and reduced paracellular transport [3][4][5]. This development of the BBB appears to be related not only to the synthesis of tight junction proteins but also to the selective expression of genes that control transport of substances between the blood and the brain [6,7]. Two families of genes that code for membrane transporters in the endothelial cell are critical to maintain brain solutes' concentration: the SoLute Carrier family (SLC transporters) and the ATP-Binding Cassette family (ABC transporters) [8][9][10]. While ABC transporters are mainly focused on the efflux of substances from the brain, in particular xenobiotics, SLC are implicated in either influx or efflux of substances. The transporters' expression pattern in the developing brain may be seen as a mid-way immature stage before a fully developed adult phenotype is achieved, nonetheless it may reflect specific needs of the developing brain i.e. BBB phenotype is adapted to each developmental stage.

Expression of efflux transporters in the epileptic tissue has been reviewed [11]and therefore the BBB is extensively studied as a potential cause of pharmacoresistance to antiepileptic drugs (AED) in animals and humans. Among the most significant modifications in the BBB phenotype, changes in tight junction proteins levels, increased expression of P-gP (P-glycoprotein, the ABCB1 gene product), MRP1 (Multidrug Resistance Protein 1, the ABCC1 gene product), and decreased expression of the monocarboxylate transporter 1 (MCT1, the product of gene SLC16A1) were found in brain tissue of patients operated on for pharmacoresistant focal epilepsies [12][13][14][15][16]. In one patient who died of status epilepticus, an increase in P-gP and MRP1 proteins was detected post-mortem [17]. Moreover, carrier-mediated efflux of valproic acid and phenytoin was observed in male adult rats [18,19], further confirming the interaction of AEDs with efflux transporters (table I) .

We hypothesize that different expression patterns of ABC and SLC transporters in the developing BBB may change brain exposure to antiepileptics. Therefore, the objective of the present study was to semi quantitatively describe the maturation of the selected ABC and SLC transporters in the developing brain of rats; additionally the data could contribute to a better understanding of antiepileptic treatment response, especially to evidence possible differences in pediatrics compared to adults. France) at 4ºC. The cerebellum, meninges, surface vessels, and brainstem were removed with forceps and the remaining tissue was finely minced with scalpels in HBSS -PSN. After centrifugation at 1500 rpm for 5min at 4ºC, the homogenized tissue was digested with 1mg/mL collagenase/dispase (Roche Diagnostics, Meylan, France) in HBSS -PSN solution, supplemented with 10 UI/μL DNAse (Roche Diagnostics, Meylan, France) and 1 μg/mL N-tosyl-lysine chloromethyl ketone (Sigma-Aldrich, St.

Experimental Section

Quentin Fallavier, France) for 1h at 37ºC. The digested tissue was homogenized by pipetting, centrifuged at 1500rpm for 5min at 4ºC, the pellet resuspended in 20% Bovine Serum Albumin (Sigma-Aldrich, St. Quentin Fallavier, France) in HBSS -PSN solution and centrifuged at 2900 rpm for 30min at 4ºC. At the end, the floating myelin layer was discarded and the pelleted microvessels were washed with Phosphate Buffer Saline -PSN (4500rpm, 5min at 4ºC) and stored immediately at -80ºC.

Total RNA extraction and RT-PCR: Total RNA from rat microvessels was extracted with TRIzol® reagent, followed by purification with RNeasy® mini kit columns (Qiagen, Courtaboeuf, France). Briefly, 20 mg of microvessels were lysed with 0.5 mL of TRIzol® reagent by pipetting.

After adding 100μL of chloroform, the mixture was centrifuged at 10000 g for 15 min at 4ºC, the clear aqueous supernatant collected, mixed with 600 μL of 70% ethanol, and loaded into the RNeasy® columns. In-column DNA elimination was performed with the RNase-free DNase set (Qiagen, Courtaboeuf, France) at room temperature. Total RNA was washed and eluted with RNase-free water (supplied with the RNeasy® kit) according to the manufacturer's protocol and stored at -80ºC. RNA concentration was measured spectrophotometrically at 260 nm in a NanoDrop® 2000c (Thermo Scientific, Villebon-sur-Yvette France), genomic DNA contamination was evaluated by measuring the A260 nm/A280 nm ratio (samples' ratios over 1.8 were considered suitable for PCR). cDNA synthesis from 0.5 μg of total RNA was performed with the RT 2 First Strand® kit (Qiagen, Courtaboeuf, France) accordingly to the manufacturer's protocol and stored at -80ºC until use.

Selection of transporters gene:

The expression of ABC efflux transporters (Abcb1a, Abcb1b, Abcg2, Abcc1, Abcc2, Abcc4, Abcc5, Abcc5and Abcc6) and SLC transporters (Slc1a1, Slc16a1, Slc22a6 and Slc22a8) involved in transport of anti-epileptic drugs was analyzed (Table I).

mRNA quantification by qPCR of cDNA: For qPCR, 1 μL of cDNA was mixed with 12.5 μL of RT 2 SYBR®green Mastermix (Qiagen, Courtaboeuf, France) and completed for 25μL with miliQwater. Mixes were loaded to customized FAST plates containing primers for the selected genes (Table 1) (CAPR 12648 customized plates, Qiagen, Courtaboeuf, France) and the qPCR reactions were performed in a 7900HT Fast Real-Time PCR System (Applied Biosystems, Villebon-sur-Yvette, France) with the following cycle conditions: 95°C for 10 min, 40 cycles of 15 sec at 95°C followed by 1 min at 60°C. Gene expression analysis was done after calculating the 2 infusion, whole blood was collected in heparinized syringes at the abdominal aorta under isoflurane anesthesia (see above), centrifuged at 2500 g for 15min at 4ºC, and plasma stored at -20ºC. Brains were collected, the cerebellum discarded, weighted and stored at -20ºC.

Digoxin, prazosin and atenolol quantification by LC/MS-MS: DGX, PRZ and ATN were quantified in brain homogenates and plasma by LC/MS-MS, using digoxin-d3 as internal standards.

Brains were first homogenized with water (2 mL per g of brain tissue) in an Ultra-Turax. Brain homogenates (400 ìL) were first treated with 1 mL methanol for protein precipitation, centrifuged at 20000 g for 15 min at 4 °C, the supernatant collected and evaporated at 40°C under N 2 current. Both brain homogenates residues and plasma samples (150 ìL) were loaded into Isolute® columns: samples were mixed with ammonia 0.75 M : methanol (4:1), loaded on to the columns and eluted with 2 mL of dichloromethane : isopropanol (7:3) followed by 2 mL dichloromethane : isopropanol (7:3) + 0.2% formic acid. After evaporation at 40ºC under N 2 current, samples were dissolved in 200 ìL of ammonium acetate 5 mM : methanol (95:5). LC/MS-MS analysis was performed in a Nexera X2

UHPLC system (Shimadzu, Noisiel, France) coupled to a triple quadrupole Finnigan TSQ Quantum Ultra mass spectrometer (Thermo Scientific, Villebon-sur-Yvette, France), using a Phenomenex RP C18 HPLC column (3 x 100 mm, 2.6 ìm). Mobile phase A consisted of ammonium acetate 5 mM and phase B of pure methanol. Runs started with 95% phase A for 1 min that decreased steadily to 5% for

Results

Differences in gene expression of the selected ABC transporters were statistically significant between P14 and P21 or P56 rats, measured as mean fold expression relatively to the housekeeping gene cyclophylin A, for the Abcb1a, Abcb1b and Abcg2 genes (Figure 1). Considering P56 rats as references for comparison with P14, the Abcb1a, Abcb1b and Abcg2 genes' relative expression calculated as the 2 -ΔΔCt (mean ± standard deviation) was respectively 0.26±0.05, 6.23±2.71 and 0.36±0.86 (Figure 2). Comparing P14 to P21 as references, Abcb1a, Abcb1b and Abcg2 genes' relative expression was 0.25±0.07, 4.07±1.91 and 0.42±0.12, respectively (Figure 3). No statistically significant difference was detected between P21 rats and P56 rats for Abcb1a, Abcb1b and Abcg2

genes. Therefore, our results showed that Abcb1a and Abcg2 gene expression appears to increase between P14 and P21 rats, while Abcb1b appeared to decrease its expression between P14 and P21 rats. Abcb1b expression appears to be substantially inferior to Abcb1a (Figure 1). In our study, we did not observe any difference between P14, P21 and P56 rats for the studied Mrp genes (Abcc1, 2, 4, 5 and 6, figure 2). Abcc6 appeared to be less expressed than the other Mrp genes. Abcc2 gene, coding for Mrp2 efflux transporter, was either undetectable or originated threshold cycles over 35.

(Place here Figures 1 and2)

SLC transporters Slc1a1, 16a1, 22a6, and 22a8 were analyzed in rat brain microvessels and data are plotted in Figure 3. A statistically significant difference was detected in the Slc16a1 gene expression, coding for monocarboxylate transporter 1 (Mct1). Slc16a1 expression in P14 rats was 2.28±0.37 folds higher than P21 rats, and 4.61±2.79 folds higher than P56 rats (Figure 2). Similarly to the ABC transporters, statistically significant differences were not observed in SLC transporters gene expression between P21 and P56 rats.

(Place here Figure 3) P-gP and BCRP efflux function were estimated by measuring K pbrain/plasma of DGX and PRZ (P-gP and BCRP substrates, respectively), ATN was used as BBB integrity marker. We observed a statistically significant lower P-gP function in P14 rats when compared with P21 or P56 rats: P14 DGX K pbrain/plasma (0.062 ± 0.015; mean ± standard deviation) was 76% and 66% higher than in P56 (0.015 ± 0.008) and P21 (0.021±0.010) respectively (figure 4). No statistically significant difference was detected in the BCRP efflux function. ATN, which is a small hydrophilic molecule that crosses the BBB only through a paracellular route (between endothelial cells) [20], was either undetected (inferior to LOQ) or originated low K pbrain/plasma values (K pbrain/plasma < 0.16) confirming BBB integrity [21].

(Place here Figure 4)

Discussion

In our study, we observed higher expression levels of the Abcb1b and Slc16a1 genes and lower expression levels of Abcb1a and Abcg2 genes in the P14 male rat specifically in brain microvessels.

Analysis of P-gP and BCRP efflux activity at the BBB reflects also post-transcriptional modifications of these genes expression and is therefore a consequence of their ontogeny.

Considering the Abcb1 genes, it is well known that they are subject to different regulatory mechanisms in their promoter region according to the tissue : Abcb1a is more expressed than Abcb1b, especially in the intestinal barrier and BBB [22][23][24], and Abcb1b is highly expressed in placenta and ovaries [25]. To study the ontogeny of the BBB and to avoid the potential influence of hormones in our results, only male rats were studied. Indeed, the expression of Abcb1b could also be up-regulated by progesterone agonists in mouse [26]. Moreover, progesterone itself may inhibit directly Abcb1b

protein [22]. Other authors identified a maximum of Abcb1b expression around P12 that decreased to adult levels, and a maximum of Abcb1a gene expression at P30 in the rat cerebellum [27]. These studies seem to corroborate our results concerning the decrease in Abcb1b and increase in Abcb1a genes expression between P14 and P56 rats specifically in brain microvessels.

P-gP efflux activity was shown in this study to be lower in P14 rats, with no difference between P21 and P56 rats, measured by the decrease in K pbrain/plasma of DGX, a specific P-gP substrate [28]. This result parallels with the observed increase in Abcb1a gene between P14 and P21 rats, which suggests a good correlation between the Abcb1a gene expression levels and P-gP activity. Abcb1b expression appears to be very low; therefore we suspect that it has not a significant impact in P-gP protein levels.

Our results show therefore a maturational increase in BBB P-gP activity during brain development.

Previous studies had reported no difference in P-gP efflux activity between immature and adult rats [29]. Nonetheless, in mice embryos, rhodamine-123 efflux coincides with the increased expression of the Abcb1a gene at embryonic day 9 [30]. Moreover, an in-vitro BBB model constructed from P14 brain endothelial cells showed an inferior P-gP efflux activity of rhodamine 123 when compared with P56 rats [31].

Little information has been published concerning the ontogeny of Mrps and Bcrp genes in the BBB.

Bcrp expression in rat brain microvessels was shown to increase after P2 [9]. The presented BCRP efflux activity results did not show maturation in BCRP activity at the BBB level, although Abcg2

gene expression significantly increased between P14 and P56. Although in-vitro studies showed an interaction of PRZ with P-gP [28], in-vivo transport studies in Abcb1a knock-out mice showed no impact of P-gP in PRZ distribution in the brain [32]. Moreover, in the present study Abcb1a also parallels Abcg2 increased expression since P14 rats until adulthood, suggesting that P-gP has not a measurable impact in PRZ efflux. Nonetheless, we cannot rule out the impact of other yet unidentified transporters in PRZ brain efflux.

Regarding Mrp1 gene expression, it was detected as highest around birth in mice cerebellum and steadily decreasing towards adult values [33]. Ontogeny data from kidney about the expression of Mrp1-7 and Mrp9 revealed an increase in the first post-natal weeks of Mrp2, 3 and 4; decrease in Mrp5 expression levels since birth and no difference in the expression of the remaining genes during development when compared with adults [34]. This study also identified Mrp5 as the major expressed Mrp in the whole brain. In an endothelial cell monolayer constructed from bovine microvessels, Mrp1, 4, 5 and 6 were detected, while Mrp2 and 3 were not observed [35]. These data seem to corroborate our results concerning the expression of Mrps in the BBB. In one study concerning MRPs protein expression in the human brain, MRP1, 4 and 5 were detected in the luminal face of brain endothelial cells and MRP2, 3 and 6 were weakly or not detected [36]. Nonetheless, Mrp6 has been observed in the human and mouse brain [37,38]. In line with this evidence, we did not detect Mrp2 possibly because we analyzed exclusively brain microvessels. A study comparing Mrp1, 2, 4, 5 and 6, and Bcrp between five mammal species (humans, rat, mouse, pig and cow), showed similar expression levels in brain endothelial cells between rats and humans, except for Mrp4 and Bcrp which presented lower expression in humans [39].

Regarding SLC genes expression, the Slc16a1 gene, coding for the MCT1 transporter, was shown to reduce its expression with brain development. MCT1 has been involved in the supply of energy substrates in the developing brain: in P10 mice, Mct1 had higher expression and lower Glut1 (glucose transporter 1, the product of gene Slc2A1) expression when compared with adults [40]. Ketone bodies are considered as an important energy source for the developing brain and they are substrates of the MCT1 transporter [41]. Apparently, MCT1 in brain endothelial cells and MCT2 (Slc16a7) in astrocytes are overexpressed in the rat brain first postnatal week in order to supply ketone bodies and other monocarboxylates down a gradient concentration [42][43][44]. Our results were in line with these results, filling the gap between the rat first post-natal week and adulthood. As previously mentioned, MCT1 expression is reduced in epilepsy. Such a reduction has been proposed as a protective mechanism to increase lactate concentrations in the brain. Increased brain lactate concentrations could have an antiepileptic action by activating the hydroxycarboxylic acid receptor 1 (HCAR1) [45].

Ketogenic diet, a procedure currently and largely used as antiepileptic therapy in children with pharmacoresistant epilepsies, induces an 8-fold increase in Mct1 expression levels in adult mice endothelial cells, mainly in the luminal endothelial membrane [46][47][48].

We did not observe any further difference in the remainder studied SLC genes between P14 and P21 rats when compared to adults. Limited information is published concerning the ontogeny of the Eaat3, Oat1 and Oat3 transporters in the brain, although Oat genes increasing expression during renal development was well characterized [49,50]. Nonetheless, the same study [33] that detected a peak Mrp1 expression in the mice cerebellum at birth, observed a high expression level of Slc22a6 that decreased to 10-20% of the adult level at P26, to further increase to adult levels around P42. Slc22a8 remained constant during development until P42 where it reached adult levels. Albeit we did not detect any statistically significant difference, our results appeared to follow the same pattern of Slc22a6 and 22a8 genes expression. Eaat3 mRNA was detected by in-situ hybridization in the human brain and was shown to increase during gestation, while after birth it maintained relatively stable values [51]. Eaat3 gene was shown to be 6 times more expressed than Eaat1 in bovine brain microvessels, where it was shown to be responsible for the efflux of glutamate towards blood [52].

This study presents information regarding the ontogeny of the selected ABC and SLC transporters but also the maturation of the two major ABC efflux transporters, P-gP and BCRP. Other factors may modify gene expression beyond age itself, either at a genome level or at a post-transcriptional level [53]. Polymorphisms in P-gP and MRP2 have been detected in patients that are pharmacoresistant [54,55]. Single nucleotide polymorphisms can change the activity or ABC and SLC genes or may modify their transduction into proteins. miRNAs were also shown to modify transduction of ABC genes [56]. In an induced mouse model of status epilepticus, there was an increase in the expression of Abcb1b in brain parenchyma and of Abcb1a in brain microvessels [57]. In mice, induction of P-gP efflux activity by AEDs was discovered [58]. Post-transcriptional regulation of Abcg2 transcripts has been shown to be mediated by micro RNAs [59,60] thus changing overall BCRP efflux activity. These observed results showed also that when animal models are used to represent human diseases or physiology, caution should be taken to control or circumvent species differences. Despite the fact that the sequential events in brain development are conserved in mammals, their time course varies accordingly to the species [61,62]. Humans accelerate brain maturation in the last trimester of pregnancy, with brain weight rate peaking in the around birth [63]. In rats, brain maturation accelerates in the first week after birth and reaches its peak by the beginning of the second post-natal week [64]. Other criteria such as early depolarizing GABA, brain glucose consumption, and the development of brain electrical activity allow considering the rat pup at the post-natal day 12-13 comparable to a full-term human newborn, post-natal day 14 to infants and post-natal day 21 to the beginning of childhood [1,[65][66][67][68].

Conclusion

Our results revealed a specific pattern of selected ABC and SLC transporters in brain microvessels of P14 rats. We speculate that other transporters involved in the transport of drugs through the BBB could also have different expression profiles in the developing brain. Moreover, differential expression of metabolizing enzymes and post-transcriptional modifications of these transporters could change brain permeability to drugs in the immature brain. Therefore preclinical studies concerning drug transport through the BBB should be promoted for drugs intended to be used in the different pediatric ages.

Abbreviations:

BBB Abcb1a ESL [69], LTG [70], OXC [69], PHT [70], PB [70] P-gP

NM_012623 Abcb1b Mrp1 NM_022281.2

Abcc1

LEV [70], PB [70] Mrp2 NM_012833.2

Abcc2

LEV [70], PB [70] Mrp4

NM_133411 Abcc4 Mrp5 NM_053924 Abcc5 Mrp6 NM_031013.1

Abcc6

Bcrp

NM_181381

Abcg2 CLB [71],LTG [72] Eaat3

NM_013032.3 Slc1a1 Oat1 NM_017224.2 Slc22a6 VPA[73] Oat3 NM_031332.1 Slc22a8 Mct1 NM_012716 Slc16a1
VPA [74] Cyclophylin A

Impact of the Antiepileptic Drugs Used in the Dravet Syndrome on the BBB phenotype in the Developing Rat Brain

The study of the ontogeny of the efflux transporters in the rat developing brain showed that the expression of some of the selected genes changed with development. To link these results with pharmacoresistance in childhood epilepsies, we decided to study the impact of the usual AEDs used in the treatment of Dravet Syndrome in this developing BBB. As previously stated, Dravet Syndrome is a good example of pharmacoresistant childhood epilepsies and we decided to study the conventional AEDs used in these children: valproic acid, stiripentol and clobazam.

We explored in this article the impact of the pharmacological treatment of the Dravet Syndrome on selected ABC and SLC genes in the microvessels of P14, P21 and P56 rats. The drugs VPA, STP or CLB were administered in these rats through sub-cutaneous osmotic minipumps that allow the continuous infusion of drug in order to obtain stable plasma concentrations. The duration of the treatment was limited to five days in order to restrict the effect of the AEDs in each class of age.

The effect of the AEDs on the expression of the selected ABC and SLC genes, in parallel to the ontogeny study previous reported, was studied by analyzing the mRNA levels of the selected genes in the microvessels of the three classes of age by semi-quantitative RT-PCR at end of the five-day treatment. We studied also the impact of VPA, STP and CLB in the efflux activity of the major ABC efflux transporters in the BBB, P-glycoprotein (P-gP) and Breast Cancer Resistance Protein (BCRP), in the three classes of age at the end of the five day treatment. This allows inputting other regulatory mechanisms that control gene expression, such as alternative splicing, mRNA stability, protein stability or direct allosteric of the AEDs inhibition or induction on P-gP and BCRP transporters.

The levels of mRNA were semi-quantified by RT-PCR in freshly isolated brain microvessels, as described for the ontogeny study. Brains of treated P14, P21 and P56 rats were collected after euthanasia of animals under deep isoflurane anesthesia. mRNA levels quantification was preformed with conventional methods on the isolated microvessels of treated rats: extraction of total RNA by the TRizol® reagent followed by purification in RNA binding columns, cDNA synthesis from total RNA and semi-quantification by real-time PCR. Recorded threshold cycles of the target gene and the control housekeeping gene (cyclophylin A, Ppia) allow calculating the 2 -ΔΔCt values.

To analyze the impact of the AEDs in the P-gP and BCRP efflux activity, brain-plasma partition coefficients (K p, brain/plasma ) were determined for digoxin (DGX) and prazosin (PRZ), a P-gP and BCRP substrate, respectively. Both molecules were administrated concomitantly through a sub-cutaneously implanted osmotic minipump in rats belonging to each of the three classes of age at the end of the five day treatment. After reaching steady-state plasma concentration, whole blood and brains were collected for dosage of DGX and PRZ by liquid chromatography coupled to mass spectroscopy (LC-MS).

The brain-plasma partition coefficients (K p, brain/plasma ) of each AED was also calculated at the end of the five-day treatment. Brain and plasma concentrations of each AED in each of the age classes were analyzed by LC-MS.

The analysis of the impact of the AEDs treatment on the developing BBB shows many interactions between drug and age, supporting the assumption that the impact of the AEDs may be modulated by the stage of brain development. VPA inhibited Abcb1a gene expression in P14 rats and induced the expression of the Slc22a6 gene in P21 rats. This impact on the Slc22a6 gene (which codes for the Oat1 transporter) is interesting because VPA is a substrate of this transporter, suggesting that it may have a role on the brain disposition of VPA. STP had effects on both ABC and SLC genes; however they were limited to P14 rats. It was identified that a single drug can modulate the BBB transporters expression pattern at a specific class of age. The results obtained after CLB treatment showed that it impacted more genes from both families and also on P56 animals. Intriguing is the fact that many genes were inhibited in P14 rats (Abcc1, Abcc5 and all SLC genes) but the expression of Abcc1, Abcc5, Slc16a1 and Slc22a6 genes were induced in P21 and P56 rats. This suggests that the regulatory mechanisms governing these genes may be differentially impacted by CLB in the developing brain.

The activity of the two major efflux transporters of the BBB was analyzed and significant interactions were also found between age and treatment. P-gP activity was induced in P14 rats but inhibited in P21

while BCRP was induced by CLB only in P21 rats. These effects did not seemed to be associated with changes of gene expression, because VPA inhibited Abcb1a gene in P14 rats and did not have any effect in P21, and CLB had no statistically significant impact on the expression of the Abcg2 gene.

This data suggests that the modulation of P-gP and BCRP activity by VPA and CLB, respectively, occurs at a post-transcriptional level, possibly a direct allosteric effect on each transporter.

This study also demonstrated age related differences in the BBB permeability of the studied AEDs during brain development. VPA and CLB appeared to have a peak in BBB permeability in P21 rats while STP BBB permeability steadily increased during brain development. Concerning STP, this result suggests unidentified transport mechanisms that regulate its brain disposition during development.

This study brings evidence that age is an important factor in the brain disposition of the AEDs used in the Dravet Syndrome. Moreover, we found significant interactions between age and treatment in both

Introduction

The hypothesis that the Blood-Brain Barrier (BBB) does play a role in the resistance to antiepileptic drugs (AEDs) in patients with epilepsy is a long-standing one 1,2 . The BBB is the special interface between the blood and the brain, composed primarily by the endothelial cells from brain capillaries that express tight junctions blocking paracellular transport 3 . These cells also express efflux transporters, belonging to the ATP-binding cassette (ABC) and SoLute Carriers (SLC) families that control the passage of substances between blood and brain 4-7 . These transporters could therefore eliminate the AEDs from the brain, and different AEDs have been identified as substrates of these efflux transporters 8 . The impact of the developing brain on these mechanisms of drug resistance in the BBB is not known. BBB phenotype is known to change with age. The increased interactions between endothelial cells and other brain parenchyma cells during development could induce a different pattern of efflux transporters, causing differences in the delivery of AEDs to the brain 9-12 . Developmental changes in the expression and protein levels of ABC transporters such as P-glycoprotein (P-gP),

Breast Cancer Resistance Protein (BCRP), Multidrug Resistance Proteins (Mrps) have already been identified 13 .

Pharmacoresistance in epilepsy is especially high in the youngest ages, with 62% of the children involved in pharmacoresistance to AEDs being less than 4 years-old 14 . Dravet Syndrome (DS) is a good model of early onset and pharmacoresistant pediatric epilepsy 15 . Seizures begin during the first year of life and continue throughout life 16 . The seizures are prolonged and often provoked by factors such as fever. The mental prognosis of these patients is poor, with a cognitive decline from normal to severe retardation as soon as the second year of life. Treatment options for this syndrome are limited [17][18][19] . Valproate is commonly used as a first-line and benzodiazepines as second line drug, but these agents are most often insufficient to prevent the recurrence of seizures. Topiramate, levetiracetam, bromide, and the ketogenic diet may provide substantial efficacy as adjunctive therapy/procedure.

Lamotrigine and carbamazepine should be avoided because they may worsen seizures. Stiripentol is the only compound which efficacy has been demonstrated in DS by a randomized controlled trial when combined with valproate and clobazam 20 . Despite of a significant improvement with these 3 drugs, the majority of patients remain with long term recurrent seizures 20 . Taken together, we hypothesized that the developing BBB could alter the delivery of AEDs to the brain when compared to the adult one. In addition, chronic treatment with AEDs could have a different impact on the BBB transporters expression pattern and efflux activity when compared to adults. In the present study we studied the brain permeability and the impact on efflux transporters of the three AEDs with an indication in DS in children (stiripentol, valproate and clobazam) in rats at three different ages of development.

their back (slightly posterior to the neck), disinfected with 70% ethanol, an incision made between the scapulae for the osmotic pump implantation and finally closed with clips.

P-gP and BCRP functionality assessment

After 5 days of treatment, rats (n=5 per group) were implanted with a second osmotic pump (Alzet ® model 2001D, 8 μL/h) containing a cocktail of DGX (12mg/kg/d), PRZ and ATN (both at 6mg/kg/d) dissolved in DMSO : polyethylene glycol (PEG) 200 . After a 4h infusion, whole blood was collected in heparinised syringes at the abdominal aorta, centrifuged at 2500g for 15min at 4ºC, and plasma stored at -20ºC. Brains were collected, the cerebellum discarded, weighted and stored at -20ºC.

Brain microvessels extraction

After the 5 day treatment (n=5, except for VPA and their controls at P21 and P56 where n=15), brains from treated and control (vehicle) rats were collected and stored in HBSS -1% PSN (HBSS Hanks Balanced Saline Solution, Life Technologies, Villebon-sur-Yvette, France; PSNpenicillin streptomycin neomycin, Sigma Aldrich, St. Quentin Fallavier, France) at 4ºC. The cerebellum, meninges, surface vessels, and brainstem were removed with forceps and the remaining tissue was homogenized with scalpels in HBSS -1% PSN. After centrifugation at 1500rpm for 5min at 4ºC, the homogenized tissue was digested with a collagenase/dispase 1mg/mL in HBSS -1% PSN solution (Roche Diagnostics, Meylan, France), supplemented with 10UI/μL DNase (Roche Diagnostics, Meylan, France) and 1μg/mL TLCK (N-tosyllysine chloromethyl ketone, Sigma Aldrich Sigma-Aldrich, St. Quentin Fallavier, France), for 1h at 37ºC. The digested tissue was homogenized by pipetting, centrifuged at 1500rpm for 5min at 4ºC, the pellet resuspended in a 20% BSA (Sigma-Aldrich, St. Quentin Fallavier, France) in HBSS -1% PSN solution and centrifuged at 2900rpm for 30min at 4ºC. The myelin layer was discarded and the pelleted microvessels were washed with PBS -1% PSN (4500rpm, 5min at 4ºC) and stored at -80ºC.

Total RNA extraction and RT-qPCR

The impact of VPA, STP and CLB separated treatments on efflux transporter gene expression was measured by mRNA levels of selected ABC and SLC transporters in isolated rat brain microvessels at post-natal days 14, 21 and 56 (P14, P21 and P56 respectively). The analysis has been performed on a series of ABC efflux transporters (Abcb1a, Abcb1b, Abcg2, Abcc1, Abcc2, Abcc4, Abcc5, Abcc5 and Abcc6) and SLC transporters (Slc1a1, Slc16a1, Slc22a6 and Slc22a8) (Table 1). Total RNA from treated and control rat microvessels were extracted with TRIzol® reagent, followed by purification with RNeasy® mini kit columns (Qiagen, Courtaboeuf, France). Briefly, 20mg of microvessels were lysed with 0.5mL of TRIzol® reagent by pipetting. After adding 100μL of chloroform, the mixture was centrifuged at 10000g for 15min at 4ºC, the clear aqueous supernatant collected, mixed with 600 μL of 70% ethanol, and loaded into the RNeasy® columns. Total RNA was washed and eluted with RNase-free water accordingly to the manufacturer's protocol and stored at -80ºC. RNA concentration was measured spectrophotometricaly at 260nm in a NanoDrop 2000c (Thermo Scientific, Villebonsur-Yvette, France), genomic DNA contamination was evaluated by measuring the A260nm/A280nm ratio (samples ratios over 1.8 are considered suitable for PCR). cDNA synthesis from 0.5 μg of total RNA was performed with the RT 2 First Strand kit (Qiagen, Courtaboeuf, France) accordingly to the manufacturer's protocol and stored at -80ºC until use. For qPCR, 1μL of cDNA was mixed with 11.5μL of 2x RT 2 SYBR®green Mastermix (Qiagen, Courtaboeuf, France) and completed for 25μL

with miliQ-water. Mixes were loaded to FAST plates (LifeTechnologies, Villebon-sur-Yvette, France) and the qPCR reactions were performed in a 7900HT Fast Real-Time PCR System (Applied Biosystems, Villebon-sur-Yvette, France) with the following cycle conditions: 95°C for 10min, 40 cycles of 15 sec at 95°C followed by 1 min at 60°C. Gene expression was calculated as 2 analysis for both P-gP and BCRP efflux activity allowed to detect significant interactions between treatment and age on P-gP efflux of VPA treated rats and on BCRP efflux activity of CLB treated rats (table 3). Age influenced all groups analyzed for P-gP efflux activity, and the changes as a function of age suggested an influence of maturation on the P-gP efflux activity. However such an influence was not detected on BCRP efflux activity of VPA treated rats unlike for the other AEDs. The impact of the treatment by each of the three AEDs on P-gP efflux activity in P14, P21 and P56 treated rats was not consistent as only VPA altered P-gp activity..A 26% decrease in P-gP efflux activity (treated 0,157 ± 0,024 vs control 0,211 ± 0,061, p<0,05) was observed in in P14 rats, and a 78% decrease in P21 rats (0,136 ± 0,041 vs 0,076 ± 0,024, p<0.05). CLB and STP had no effect on BBB P-gP function. BCRP efflux activity in control and treated rats in P14, P21 and P56 rats (Figure 5). CLB induced 5,6-fold the BCRP activity exclusively in P21 rats (PRZ K p,brain/plasma 0,292 ± 0,05 in treated animals vs 1,629 ± 0,380 in controls, p<0.001). BCRP activity was not affected by the chronic treatment with either VPA or STP. ATN K p,brain/plasma which we studied as a marker of BBB integrity was always under 0,1 or brain concentrations were below the limit of quantification in brain homogenates (1 ng/mL), confirming the experimental BBB integrity in every group studied (data not shown).

VPA, STP and CLB permeability in the developing brain

BBB permeability to VPA, STP and CLB measured by the brain/plasma partition coefficient (K p,brain/plasma ) in P14, P21 and P56 rats (fig 1). For VPA (Fig. 6A), a statistically significant difference in the VPA brain permeability was observed between P21 and P56 rats, 0.09 ± 0.03 vs 0.06 ± 0.01, respectively (p<0.05, Kruskal-Wallis test), although there was no difference between P14 and P56 rats. K p,brain/plasma values ranged between 0.05 and 0.1. STP, K p,brain/plasma increased from 0.21 ± 0.02 at P14, 0.38 ± 0.07 at P21, to 0.62 ± 0.10 in P56 (Fig. 6B). A K p,brain/plasma increase was observed for CLB from 2.82 ± 0.355 in P14 rats to 3.37 ± 0.307 in P21, and finally a decrease to 2.56 ± 0.252 in P56 rats (Fig. 6C).

Discussion

The present work reported for the first time the impact of the AEDs used to treat DS on the expression of surface transporters in the developing BBB.

Three age-classes were selected in order to compare the developing brain in rats and in humans and specifically analyzed brain microvessels. We considered P14 rats as representative of infants, P21 and children and P56 of adult humans.

The potential impact of the studied AEDs in the efflux activity of the two major efflux transporters in the BBB, P-gP and BCRP, was investigated as well as the changes in the brain disposition of the selected AEDs during brain development. As the rat brain develops rapidly, in order to assign the observed results to each corresponding age-class in the immature rat, we limited the treatment with each AED to five days. This duration of treatment was considered sufficient to achieve steady-state because of the continuous delivery of the AEDs by the osmotic minipumps, which minimizes the effect of the short half lives of the AEDs reported in immature rats. The observed changes in the expression and function of the studied ABC and SLC transporters retrospectively supported the assumption that this duration of treatment was sufficient.

The results on VPA brain disposition showed a decrease in VPA K p,brain/plasma between P21 and P56 rats. The observed values of K p,brain/plasma ranging between 0.05 and 0.1, although slightly lower than previously reported, are in the same range 21,22 . In one study comparing the in-vivo influx (blood-tobrain) of VPA in P14 and P56 rats and influx (apical-to-basolateral) in an in-vitro BBB model constructed with cells from P14 and P56 rats, VPA was shown to have a higher influx in P14 rats 23 .

Although our results did not show any significant difference between P14 and P56 rats, we presented data as partition coefficients after 5 days of treatment which reflect not only the possible modulation of the BBB transporters but also the equilibrium between influx and efflux of VPA through the BBB unlike in this previously reported study.

inducing the expression of Abcc1, Abcc5, Slc16a1, Slc22a6 and Slc22a8. We apparently identified an AED whose effects on the BBB, particularly on transporters who are responsible for the elimination of xenobiotics, is limited to very young animals. This has not been previously reported using STP as a substrate of a transporter.

The permeability of CLB in the developing brain seems to reach a maximum in P21 rats. We observed a 5.6-fold reduction in the BCRP efflux activity in P21 rats treated with CLB. Although CLB seems to be a substrate of BCRP 21 , because of the high brain permeability of CLB this possible self-inducted efflux of the brain mediated by BCRP may be attenuated. We did not quantify the active metabolite Ndesmethylclobazam (nCLB) in the rat brain because in all samples it was either undetectable or under the limit of quantification (LOQ nCLB = 32 ng/mL). A previous report showed that metabolism of CLB in rats may be different from humans: although nCLB is produced in the rat liver, its concentration is lower than in humans 37 . Moreover, another study in rats and mice showed that nCLB is detected in mouse brain but not in rats 38 . Our results suggest therefore that nCLB may have a minimal impact on seizures when using rats as model for epilepsy.

The expression of transporters in the developing BBB appears to be highly influenced by CLB treatment, as shown by our results. All studied SLC genes were inhibited in P14 rats plus Abcc1 and Abcc5. Abcb1a however was induced in P14 rats although this result did not have apparently any influence on P-gP efflux activity. CLB is actively eliminated from the brain by BCRP 21 and we observed a statistically significant increase in BCRP efflux of PRZ in P21 rats. This lead us to suggest that CLB induce its own elimination by BCRP from the brain in P21 rats through a mechanism that is not dependent of BCRP gene expression regulation. To our knowledge, CLB has not been identified as a substrate of other ABC or SLC transporters. The effects on Slc1a1 gene, which codes for the glutamate aspartate transporter (GLAST), may be relevant because this protein transports glutamate, an aminoacid which has excitatory effects on synapses 39,40 . The increase in GLAST expression, as observed in P21 and P56 rats brain microvessels, had already been observed in the hippocampus of adult rats treated with CLB, who had seizures induced by amygdalar injection of Fe 3+41 . The observed reduction in SLC genes expression by CLB exclusively in P14 rats confirmed our hypothesis that the developing BBB responds differently at different ages. Expression of Abcc1 and Abcc5 genes were induced by CLB in P56 rats, confirming the different sensibility of the developing BBB.

As previously mentionned, Slc16a1 gene is involved in the supply of energy substrates to the brain.

This gene has also been linked to epilepsy because it was observed to be inhibited in human brain microvessels obtained from temporal lobe epilepsy as well as in animal models of epilepsy 42,43 . In P14 rats STP induced its expression while CLB reduced its mRNA levels. Although we cannot predict the overall effect of these two AEDs when they are administered together in DS, these results suggest however that Mct1 in brain microvessels may have an important role in epilepsy.

Abcc2 gene expression was very low or virtually not detected in the brain rat microvessels at any age.

In previous studies, Mrp2 also showed limited expression in the brain 44,45 . However, MRP2

polymorphisms were associated with pharmacoresistance in epilepsy patients and phenytoin seemed to be a substrate of this transporter 46,47 suggesting that the rat should not be used as an animal model for studies on Mrp2.

Conclusion

Chronic administration of the AEDs on the developing rat BBB had different impacts according to age on the expression of the selected ABC and SLC genes and also on the efflux activity of P-gP and BCRP. We also observed age-related differences in the brain permeability of VPA and STP.

Replication of these experimental procedures in an animal model of the DS could bring additional informations by analyzing the influence of the disease in the developing BBB. Also the impact of these three drugs in combination should be analyzed, as they are administered to children together and potential drug-drug interactions may occur at the developing BBB. and BCRP substrates respectively and calculation of brain/blood partition coefficients (Kp, brain) in steady-state conditions. For gene expression analysis, rat brain microvessels were extracted and the mRNA levels were semi-quantified by qRT-PCR. Data were analyzed by 2-way ANOVA followed by Bonferoni-corrected t-student post-hoc analysis and considered significant if P ≤ 0.05.

Results: After 5 days, digoxin Kp (mean±SD) increased from 0.070±0.01 to 0.136±0.016 in P21 rats and from 0.042±0.02 to 0.077±0.07 in P56, both differences being statistically significant (P < 0.01).

A trend to decrease prazosin Kp (0.467±0.057 vs. 0.370±0.023) was noticed in P21 but was not statistically significant. Although P-gP and BCRP mRNA levels were significantly higher in P56 than in P21 rat brain microvessels (P-gP P < 0.01, BCRP P < 0.05), VPA treatment did not change both genes expression. Nonetheless, we observed a statistically significant 4-fold increase (2-DDCt) in the expression of Slc22a6 gene (OAT1, organic anion transporter 1) after VPA treatment in P21 rats (P < 0.01), suggesting possible interaction between VPA and OAT1.

Impact of carbamazepine on the pattern of ABC and SLC transporters of the developing BBB

In addition to drugs used in Dravet syndrome, the impact of CBZ on the pattern of ABC and SLC transporters of the developing BBB was also studied. Rats at P14, P21 and P56 were treated with 50mg/Kg/day for 5 days, as previously described for VPA, STP and CLB.

Considering the impact on the expression of ABC and SLC genes, significant interactions with age were in Abcb1b, Abcc1, and Slc16a1 genes (Table 5). The effect of age in the expression of the Slc16a1 gene was still statistically significant despite an interaction was found. Concerning the impact of CBZ in P-gP and BCRP efflux activity, age was a significant factor influencing P-gP and BCRP activity (Table6), however it was not detected any significant interaction of CBZ with these transporters (Figure8). CBZ effects on the developing BBB were limited to an increase in expression of the Abcb1b gene.

Although this gene also codes for P-gP in rodents, its expression is very limited in the brain (Cui et al., 2009). Moreover, no impact was observed in either P-gP or BCRP efflux activities and CBZ appeared not to be a substrate of either P-gP or BCRP [START_REF] Ambroziak | Exposure to antiepileptic drugs does not alter the functionality of P-glycoprotein in brain capillary endothelial and kidney cell lines[END_REF][START_REF] Cerveny | Lack of interactions between breast cancer resistance protein (bcrp/abcg2) and selected antiepileptic agents[END_REF][START_REF] Fortuna | Evaluation of the permeability and P-glycoprotein efflux of carbamazepine and several derivatives across mouse small intestine by the Ussing chamber technique[END_REF]Zhang et al., 2011). The increase in expression of Abcc1 and Abcc5 by CBZ in P56, which parallels the observations in treatment with CLB, suggested that these transports are also important in the adult BBB. Although these results did not allowed to establish causal relationships between the effects of CBZ in the developing BBB and pharmacoresistant to AEDs in children, this drug seemed to have a lower impact in the pattern of ABC and SLC transporters in the developing BBB.

Interaction of VPA with the efflux transporters P-gP and BCRP in an in vitro model of the developing BBB constructed with primary cultures of rat brain endothelial cells

The results obtained in the previous section concerning the impact of VPA in the in vivo efflux activity of P-gP suggested an interaction of VPA with P-gP at a post translational level in the developing BBB.

Therefore, to study this interaction at the cellular level, the construction of an in vitro BBB model with primary cultures of rat brain endothelial cells seemed a good strategy. Previous studies developed with an in vitro model of the adult BBB were the starting point for the development of the immature model (Harati et al., 2013;[START_REF] Josserand | Evaluation of drug penetration into the brain: a double study by in vivo imaging with positron emission tomography and using an in vitro model of the human blood-brain barrier[END_REF][START_REF] Lacombe | In vitro primary human and animal cell-based blood-brain barrier models as a screening tool in drug discovery[END_REF][START_REF] Mabondzo | Validation of in vitro cell-based human blood-brain barrier model using clinical positron emission tomography radioligands to predict in vivo human brain penetration[END_REF][START_REF] Megard | A co-culture-based model of human blood-brain barrier: application to active transport of indinavir and in vivo-in vitro correlation[END_REF], and P21 rats were selected for in vitro modeling of the immature BBB. This in vitro model was constituted by a co-culture of P21 rat brain endothelial cells with a primary culture of P2 rat glial cells.

Endothelial cells were seeded in the upper side of a Transwell insert ®, which recreates blood compartment (luminal or apical compartment), while glial cells were seeded in the bottom of a 12-well plate, mimicking brain parenchyma (abluminal or basolateral compartment) (Figure 8). In order to validate the in vitro BBB model constructed with primary cultures of adult and immature rat brain endothelial cells, the TEER of Transwell Inserts® was followed every other day when media were changed. Peak TEER was observed at the 6 th day of co-culture with mean TEER 270 Ω.cm 2 (CV% = 6.5%, n = 6) for the immature model and at the 10 th of co-culture, with mean TEER 258 Ω.cm 2 (CV% = 4.7%, n = 6) for the adult model. These TEER values are above the 150 Ω.cm 2 , suggesting that a confluent monolayer with restrictive paracellular transport was formed in both in vitro BBB models (adult and immature).

The restrictive paracellular transport in the in vitro model of the BBB was analyzed by calculating the The apparent discrepancy between the effects of VPA on the expression of Abcb1a and on the P-gP DGX efflux activity might be related to post-transcriptional modulation of the Abcb1a gene product.

These results from the in vitro BBB model suggest that inhibition of P-gP, as observed in the immature and adult BBB model, appears to be made at the transporter level in the endothelial cell membrane. This seems to substantiate the observed induction of in P-gP efflux by VPA in P21 rats (see section 5.3). P-gP is known to have a biphasic behavior: the P-gP mediated efflux of phenobarbital and rhodamine 123 were shown to be induced at low doses of verapamil but rather inhibited when verapamil concentrations increase [START_REF] Yao | Verapamil exerts biphasic modulation on phenobarbital transport across the blood-brain barrier: evidence from an in vivo and in vitro study[END_REF]. Moreover, recent studies showed that in in vitro models of the BBB constructed with transformed cell lines of brain endothelial lineage (RBE4 and hCMEC/D3), VPA inhibited P-gP activity at therapeutic concentrations but this effect was lost at higher concentrations ([VPA] = 300 μM, log[VPA] = 2.48) [START_REF] Alms | Marked differences in the effect of antiepileptic and cytostatic drugs on the functionality of P-glycoprotein in human and rat brain capillary endothelial cell lines[END_REF]. These results seem to be in line with the observations in P-gP mediated VBL efflux in the in vitro BBB model of primary endothelial cells from immature rats. This further suggests that the impact of VPA on the efflux activity of P-gP is rather exerted at a post-transcriptional level.

Discussion

This work was developed to bring more information onthe potential role of the developing BBB in the emergence of resistance to AEDs in the pediatric population. As previously analyzed in the introduction to this work, pharmacoresistance in childhood epilepsies is still a major concern in neuropediatrics.

The experimental work was initiated by establishing two main hypotheses:

1 -The developing BBB may have a pattern of membrane transporters different from the adult one that could explain pharmacoresistance in epileptic children;

2 -The impact of the AEDs used to treat the Dravet Syndrome on the developing BBB pattern of membrane transporters may be different from the mature BBB.

Discussion on the methodology used

The Dravet syndrome was chosen as a model for pharmacoresistant epilepsies in childhood because it is a syndrome that first appears during the first year of life, when the brain is developing. This syndrome, as discussed in the introduction, is a prototypical childhood epileptic syndrome where pharmacoresistance is frequent. Moreover, the frequent seizures that are later followed by impairment of cognitive and motor development make relevant the study of pharmacoresistance in this syndrome.

The majority of children with this syndrome are treated with the AEDs VPA, STP and CLB.

Nonetheless it is a form of severe epilepsy that frequently does not respond to treatment and surgery cannot be a therapeutic option because children develop generalized seizures.

The study of pharmacoresistance in human subjects is of difficult implementation due to ethical concerns, especially when referring to the pathologies of pediatric populations. In this case of pharmacoresistance in epilepsy due to the developing BBB, non invasive techniques such as PET-scan studies with specific markers may be useful. These studies are however sparse in the published literature concerning the pediatric populations and are not without disadvantages, in this case the manipulation of radioactive material can be deleterious to the developing brain. The use of developing animals appeared to be a better strategy to analyze the role of the developing BBB in the pharmacoresistance to the AEDs. The rat was chosen because there are already published studies on the development of the BBB in this species, young animals develop rapidly which permits to obtain results in a short time, and also many in vitro BBB models have been published with endothelial cells from rat brains. However data from human tissue would greatly increase the usefulness of these results. Although it is very difficult to study the ontogeny of the ABC and SLC genes in healthy tissue due to ethical concerns, the comparison between these genes expression in brain tissue obtained from children and adults submitted to epilepsy surgery may unravel differences between the developing and mature human BBB. It must however be kept in mind that epilepsy surgery is performed in a restrict set of epilepsies, such as focal epilepsy.

The ontogeny of the ABC and SLC efflux transporters was studied in P14, P21 and P56 rats. These age classes were selected because they correspond to human infants, children and adults: this allowed the study of the impact of the Dravet syndrome treatment in the affected pediatric ages. The genes were selected by their relevance in pharmacoresistance or their potential interaction with AEDs. The ontogeny of the ABC and SLC efflux transporters was studied by measuring the mRNA levels of the selected genes. mRNA levels however do not take into account the possible post-translational mechanisms (examples: alternative splicing or mRNA stability) that regulate protein levels. Analysis of the protein levels of these transporters by Western blot could have been useful to understand if regulation of these genes expression may actually change the actual functional protein levels in the endothelial cell membrane. Nonetheless, the activity of the major efflux transporters of the BBB was studied by analyzing the efflux of known substrates (DGX for P-gP and PRZ for BCRP). This analysis of efflux activity allowed the integration of transcriptional and post-transcriptional modulation on the transporters. This study was limited to P-gP and BCRP because substrates for the other ABC and SLC transporters have not been well characterized.

Regarding the second objective, to study the impact of the AEDs used in the Dravet syndrome on the developing BBB these drugs were administered by subcutaneous implantation of osmotic pumps.

These pumps allow the convenient infusion of the drug throughout the duration of study and the achievement of stable concentrations in plasma and brain. This allowed direct calculation of the K p,brain/plasma values for each AEDs after measuring plasma and brain concentrations. The treatment with the AEDs was limited to five days because collection of results could be biased: starting treatment in P14 rats and maintaining it for instance 7 or more days would lead to the collection of data in brains at P21.

The in vitro model of the developing rat BBB was constructed with primary cultures of brain endothelial cells. This type of cells was used because they could reflect more closely the developing BBB phenotype. This model however needs to be further validated in order to assure that it reflects the pattern of efflux transporters in in vivo, for example by selecting substances that are substrates of the transporters and correlating the in vivo permeability with the in vitro results. This method was nonetheless studied in regard to its reduced paracellular transport, measured by the sucrose permeability, and the P-gP efflux activity, measured by the vinblastine efflux ratio.

Discussion on results

Animal models of immature blood brain barrier for optimizing drug disposition

The published available information concerning the cellular constituents of the BBB were analyzed and also their impact in the sustainability of the BBB properties. It was also analyzed the mechanisms through which cell-cell interactions in the brain parenchyma induce the BBB phenotype in newly formed endothelial cells and how these pathways in the developing brain could change the pattern of membrane transporters in the brain endothelial cells. The compilation of the published in vitro BBB models using primary cultures of brain endothelial cells showed that more than half of the selected studies did not mention the age of the animals whose brain endothelial cells were isolated. Imprecise terms such as "calves" (for bovine cells) or age ranges (1-3 months) were used. The only study that specifically addressed differences between immature and adult BBB (interestingly using VPA as molecular marker) is to a certain extent not adequate due to the low confluence of the endothelial cell monolayer. Therefore, more information on the potential differences between the adult and the developing BBB were needed to develop a more robust BBB model, because drug transport through the BBB can be influenced by the existence of efflux and influx transporters in the endothelial cells.

Following the finding that until today only one in vitro model was constructed to analyze differences in the permeability between the immature and adult brain, we proceeded with a study of the ontogeny of selected ABC and SLC genes in the microvessels of the immature rat brain 6.2.2. Ontogeny of ABC and SLC transporters in the microvessels of developing rat brain.

The differences in the expression of selected ABC and SLC genes in the microvessels of the developing rat brain were analyzed at different ages of maturation. The expression of most of the major efflux transporters of the BBB, P-gP and BCRP, was shown to increase during brain development: Abcb1a and Abcg2 genes increased their expression while Abcb1b decreased its expression. Furthermore, the efflux activity of P-gP was shown to increase during brain development.

Regarding SLC genes, the monocarboxylate transporter 1 (Mct1), the product of the Slc16a1 gene, also decreased during development of the rat brain. These genes are of relevance because they seem to be responsible for the transport of AEDs: ESL, LTG, OXC, PB and PHT are substrates of P-gP (Luna-Tortós et al., 2008;Zhang et al., 2011); LTG and CLB are substrates of BCRP (Nakanishi et al., 2013); and VPA is a substrate of MCT1 (Adkison and Shen, 1996;Fischer et al., 2008). Furthermore MCT1 is transporter that was found to be repressed in epileptic tissue of patients who underwent brain surgery for pharmacoresistant focal epilepsy (Lauritzen et al., 2011(Lauritzen et al., , 2012). These differences may therefore have an impact on the brain distribution of the AEDs that are substrates of these transporters, and may also be related to potential drug-drug interactions with other intentionally administered drugs that modulate these transporters.

The results obtained in this study showed differences in the expression and function of ABC and SLC genes during BBB maturation. This led us to investigate the additional role of the pharmacological treatment of the Dravet Syndrome as another factor in the modulation of the BBB transporters phenotype.

Impact of the antiepileptic drugs used in the Dravet syndrome on the

Blood-Brain Barrier in the immature rat brain

The impact of the drugs usually used to treat Dravet Syndrome, VPA, STP and CLB, on the developing BBB was analyzed. When considering VPA treatment, Abcb1a gene expression was inhibited in P14 rats and Slc22a6 gene expression was increased in P21 rats. These results show a differential impact according to age and the studied gene after treatment with VPA. In addition, P-gP efflux activity was induced in P14 rats but inhibited in P21 rats. The impact on the efflux activity of P-gP shows that VPA modulates P-gP at a post-transcriptional level, as the differences observed in Abcb1a gene expression could not explain the changes in P-gP efflux activity. Interestingly, as VPA is substrate of OAT1 (the product of gene Slc22a6), the increase in this gene expression may have an impact on VPA distribution to the brain. We observed a higher VPA permeability at P21 rats suggesting that OAT1 expression may influence VPA brain permeability but more information on the role of OAT1 in the BBB is needed.

The only drug that is approved for the treatment of the Dravet Syndrome, STP, influenced exclusively in P14 rats the expression of some ABC and SLC genes. This showed that the BBB responded differently according to the brain development stage and the regulatory mechanisms governing gene expression were apparently only influenced by STP in P14 rats. Moreover, the steadily increase in STP brain permeability suggested a yet unidentified mechanism that could be responsible for the STP transport at the BBB that only became fully operational in the adult brain.

Finally, CLB was responsible for a more important impact on the expression of both ABC and SLC genes, either in the developing or the adult BBB. We identified differential impacts of this drug according to age: while Abcc1, Abcc5 and Slc1a1 genes were inhibited in P14 rats, in P21 or P56 these genes were induced. These results suggest that the mechanisms underlying the expression of these genes are differently susceptible to CLB according to age. In parallel with the observed results for VPA, the induced BCRP efflux activity observed in P21 rats after the chronic treatment with CLB were not consistent with changes in Abcg2 gene expression. This suggested once more that the changes in the efflux activity were induced at a post-translational level. Because CLB seemed to be a substrate of BCRP, the induction of BCRP efflux activity that could reduce CLB brain disposition was apparently compensated by the high brain partition coefficient of CLB.

CBZ was studied in addition to the AEDs used in the treatment of the Dravet syndrome. CBZ induced limited changes in the pattern of ABC and SLC transporters of the developing BBB: only Abcb1b gene was impacted in developing rats. CBZ did not have any impact in the efflux activity of P-gP and BCRP, suggesting that the impact of CBZ in the BBB were limited. The use of this immature BBB in vitro model showed that the interaction of VPA with P-gP observed in vivo appeared to be caused by a direct effect on P-gP transporter. The efflux activity of P-gP appeared to be inhibited at low concentrations of VPA however higher concentrations seemed to lose this inhibitory effect. These results were in accordance with previous published studies,

The results observed in these works showed a multitude of interactions between brain development and AEDs treatment. This work indicates that the impact of the treatment with the AEDs used for the Dravet syndrome on the pattern of efflux transporters may be influenced by the developing BBB.

Interactions were observed not only for the activity of the major BBB efflux transporters, but also on the expression of the ABC and SLC genes. Moreover, these changes may have an impact on the brain disposition of the studied AEDs.

Perspectives

We showed that development has an impact in the pattern of efflux transporters in developing BBB and changes are more pronounced in the youngest animals. As perspectives, the analysis of other age classes, such as P10 rats that more closely resemble newborn humans or P35 rats that are approximants of adolescent animals, could further evidence differences in the developing BBB. In adition, the ontogeny of the ABC and SLC genes could be analyzed in already available animal epilepsy models, either genetically modified animals such as for Dravet syndrome or tuberous sclerosis; or induced epilepsy models such as the kindled rat induced by administration of pilocarpine or kainic acid.

This experimental strategy should be applied in rats treated concomitantly with the three AEDs used for the Dravet Syndrome. This could unravel the possible interactions of these drugs at the level of the developing BBB. Furthermore, the existence of an animal model of the Dravet Syndrome could be of particular interest: the potential different impact on the developing BBB of non-healthy animals could be more representative of the actual human patients. More specific work should be developed to study the molecular mechanisms of interaction between VPA and P-gP, and CLB and BCRP, for instance by using an in vitro model of the developing BBB. The role of the OAT1 transporter on the brain disposition of VPA should be made clearer, as this transporter has a wide range of substrates and could be responsible for significant drug-drug interactions with VPA. Finally, the potential existence of transporters involved in the brain disposition of STP should be addressed in order to understand the observed steady increase in STP brain permeability.

Data from human tissue would greatly increase the usefulness of these results. Although it is very difficult to study the ontogeny of the ABC and SLC genes in healthy tissue due to ethical concerns, the comparison between these genes expression in brain tissue obtained from children and adults submitted to epilepsy surgery may unravel differences between the developing and mature human BBB. It must however be kept in mind that epilepsy surgery is performed in a restrict set of epilepsies, such as focal epilepsy.

Conclusion

The two hypotheses that oriented this work were first the possibility that the developing BBB presents a different pattern of efflux transporters, and second that the impact of the AEDs treatment on this pattern of efflux transporters may be influenced by the developing BBB. The experimental strategy developed in this work showed that indeed the BBB pattern of efflux transporters is different between the developing and the adult brain, especially for the two major transporters present in the BBB, P-gP and BCRP. Furthermore, the complexity of interactions observed between brain development and the AEDs used in the treatment of the Dravet Syndrome, that seem to corroborate our second hypothesis,

shows that age and treatment must both be taken in account when studying the role of the BBB in the

  Paris Descartes PRES Sorbonne-Paris-Cité Titre: Etude du Passage des Médicaments Antiépileptiques à travers la Barrière Hémato-Encéphalique Etudiant : Ricardo VIANA SOARES Directeurs de Thèse: Pr. Gérard PONS and Dr. Aloïse Mabondzo Equipes d'Accueil: 1 -U 1129 Inserm/Université Paris-Descartes/CEA (Epilepsies de l'Enfant et Plasticité Cérébrale) 149 rue de Sèvres, 75015 Paris, France 2 -CEA/DSV/iBiTEC-S/SPI/LEMM (Laboratoire d'Etudes du Métabolisme des Médicaments) CEA de Saclay, 91191 Gif-sur-Yvette Résumé:

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. Types of transport through the BBB Figure 2. Types of SLC transporters Figure 3. Phases of BBB development

Figure 4 .

 4 Figure 4. Development of brain vasculature between embryonic day 9.75 (E9.72) and E10.5

Figure 5 .Figure 6 .

 56 Figure 5. Effects of CBZ on the expression of ABC (A) and SLC (B) efflux transporters genes in the microvessels of P14, P21 and P56 rats Figure 6. Impact of the CBZ treatment on P-gP efflux activity measured by Kp,brain/plasma of DGX in post-natal day 14 (P14), 21 (P21) and 56 (P56) rats.

Figure 7 .

 7 Figure 7. Impact of the CBZ treatment on BCRP efflux activity measured by Kp,brain/plasma of PRZ in post-natal day 14 (P14), 21 (P21) and 56 (P56) rats.

Figure 8 .Figure 9 .

 89 Figure 8. Schematic representation of the in vitro blood-brain barrier model Figure 9. Interaction of VPA with P-gP (A) and BCRP (B) in an in-vitro model of BBB

P14

5. 1 -

 1 Resulsts of the exhaustive review of the published in vitro BBB models This work is to be submitted to "Moelcular Pharmaceutics" journal under the tittle: "In vitro models of the immature blood brain barrier for optimizing drug disposition" List of authors: R. Soares, A. Mabondzo, C. Chiron, S. Chhun, G. Pons 5.2 -Ontogeny of the ABC and SLC transporters in the microvessels of developing rat brain This work was submitted to "Fundamental and Clinical Pharmacology" on the 15 th of June 2015 under the title: "Ontogeny of the ABC and SLC transporters in the microvessels of developing rat brain" List of authors: Ricardo V. Soares, Tuan M. Do, Aloïse Mabondzo, Gérard Pons, Stéphanie Chhun 5.3 -Impact of the Antiepileptic Drugs used in the Dravet Syndrome on the Blood-Brain Barrier in the Immature Rat This work is to be submitted to "Molecular Pharmaceutics" journal under the title: "Impact of the Antiepileptic Drugs used in the Dravet Syndrome on the Blood-Brain Barrier in the Immature Rat" List of authors: Ricardo Viana Soares, Stéphanie Chhun, Vincent Jullien, Tuan Minh Do, Anne-Cécile Guyot, Catherine Chiron, Gérard Pons, Aloïse Mabondzo The results assembled in this section have been partly presented as an oral communication at the 11 th European Congress on Epileptology in july 2014 in Stockholm, Sweden; as an oral communication at the 19 th Annual Meeting of the French Society of Pharmacology and Therapeutics in april 2015 in Caen, France; and as a poster in 11 th International Conference on Cerebral Vascular Biology in june 2015 in Paris, France.

Figure 1 .

 1 Figure 1. Types of transport through the BBB (Abbott et al., 2010a), adapted.

Figure 2 .

 2 Figure 2. Types of SLC transporters[START_REF] Hediger | The ABCs of solute carriers: physiological, pathological and therapeutic implications of human membrane transport proteinsIntroduction[END_REF]

Figure 3 .

 3 Figure 3. Phases of BBB development. Endothelial cells (EC) start to sprout following a Vascular Endothelial Growth Factor (VEGF) gradient produced by Neuropercursor cells (NC). Differentiation and maturation of the BBB further proceeds by increasing interactions with pericytes (PC), astrocytes (AC), microglia (MG) and neurons (N) (Liebner et al., 2011b), adapted.

Figure 4 .

 4 Figure 4. Development of brain vasculature between embryonic day 9.75 (E9.72) and E10.5[START_REF] Fantin | The embryonic mouse hindbrain as a qualitative and quantitative model for studying the molecular and cellular mechanisms of angiogenesis[END_REF].

BBB functioning 2 -

 2 To study the ontogeny of the ABC and SLC genes in the developing BBB using the rat (Rattus norvegicus) as model 3 -To study the impact of the chronic administration of the Dravet syndrome treatment (valproic acid, stiripentol and clobazam) to developing rats on the expression and function of the ABC and SLC efflux transporters. 4 -To study the interaction of the AED valproic acid with the efflux transporters P-gP and BCRP in an in vitro model of the developing BBB constructed with primary cultures of rat brain endothelial cells.

  which level does not change during the experimental procedure. The end-point was the 2 -ΔCt values of each gene, where Δ Ct is the difference between the threshold cycle of the target gene and the housekeeping gene cyclophylin A (Δ Ct = Ct target gene -Ct housekeeping gene ). Threshold cycle refers to the cycle in the polymerase chain reaction whose measured fluorescence signal first differs from background noise.

1 ,

 1 Mct1, Mct2 orEaat3; and most importantly for the major efflux transporters of the BBB: P-gP and Bcrp. We speculate that the expression and function of more transporters can be regulated in an age-dependent matter in the BBB and therefore in vitro BBB models should be developed specifically for studies in the pediatric population. This may also be very useful for the study of the pharmacoresistance in childhood epilepsies because little is known about the role of the developing BBB in the resistance to the AEDs. Article:" In vitro models of the immature blood brain barrier for optimizing drug disposition" (To be submitted to "Molecular Pharmaceutics" List of authors: R. Soares 1,2 , A. Mabondzo 2 , C. Chiron 1 , S. Chhun 3 , G. Pons 2 1 -Inserm U1129, Paris, France; University Paris Descartes; CEA, Gif-sur-Yvette, France 2 -CEA, Direction des Sciences du Vivant, iBiTec-S, Service de Pharmacologie et d'Immunoanalyse, 91191, Gif-sur-Yvette, France 3 -Inserm U1151, INEM, Paris, France; University Paris Descartes. Hôpital Necker-Enfants Malades, 149 rue de Sèvres 75015 Paris, France) In vitro models of the immature blood brain barrier for optimizing drug disposition R. Soares 1,2 , A. Mabondzo 2 , C. Chiron 1 , S. Chhun 3 , G. Pons 2 1 -Inserm U1129, Paris, France; University Paris Descartes; CEA, Gif-sur-Yvette, France 2 -CEA, Direction des Sciences du Vivant, iBiTec-S, Service de Pharmacologie et d'Immunoanalyse, 91191, Gif-sur-Yvette, France 3 -Inserm U1151, INEM, Paris, France; University Paris Descartes. Hôpital Necker-Enfants Malades, 149 rue de Sèvres 75015 Paris, France)

5. 2 .

 2 Ontogeny of ABC and SLC transporters in the microvessels of developing rat brain Brain development is a continuous process of human development since the formation of the neural tube until the adult stage. Blood-Brain Barrier (BBB) also develops since the first stage of angiogenesis towards the final mature adult state. This development of the BBB is considerably influenced by the secretion of paracrine substances, such as Vascular Endothelial Growth Factor (VEGF) that are secreted by neuropercursor cells. The BBB hallmarks are the existence of tight junctions between endothelial cells and the expression of efflux transporters from the ATP-binding cassette transporters (ABC) and the SoLute Carriers (SLC) transporters. These transporters have a fundamental role in the disposition of nutrients and xenobiotics in the brain parenchyma.

Running Title :

 : Efflux transporters ontogeny in rat BBB Ricardo V. Soares a,b , Tuan M. Do b , Aloïse Mabondzo b , Gérard Pons a , Stéphanie Chhun c * a -Inserm U1129, Paris, France; University Paris Descartes; CEA, Gif-sur-Yvette, France b -CEA, Direction des Sciences du Vivant, iBiTec-S, Service de Pharmacologie et d'Immunoanalyse, 91191, Gif-sur-Yvette, France c -Inserm U1151, INEM, Paris, France; University Paris Descartes. Hôpital Necker-Enfants Malades, 149 rue de Sèvres 75015 Paris, France * -Corresponding Author Corresponding author address: UMR 1151, INEM, Hôpital Necker-Enfants Malades, 149 rue de Sèvres 75015 Paris, France Email: stephanie.chhun@nck.aphp.fr Phone number : 0033144495343

Figure 1 .

 1 Figures:

Figure 2 .

 2 Figure 2. Ontogeny of selected ABC and SLC transporters in isolated rat brain microvessels at postnatal days 14, 21 and 56 (P14, P21 and P56 respectively). 2 -ΔΔCt values (ΔΔCt = ΔCt immature -ΔCt adult ) are plotted and statistical analysis was performed by One-way analysis of variance (**p<0.01,

Figure 3 .

 3 Figure 3. Ontogeny of selected SLC transporters in isolated rat brain microvessels at post-natal days 14, 21 and 56 (P14, P21 and P56 respectively). 2 -ΔCt values (ΔCt = Ct target -Ct Ppia ) are plotted in a semi-log scale and statistical analysis was performed by One-way analysis of variance (**p<0.01,

Figure 4 .

 4 Figure 4. P-gP efflux (A) and BCRP efflux (B) measured by DGX and PRZ K p,brain/plasma , respectively at steady-state, plotted as mean ± standard deviation , in post-natal days 14, 21 and 56 (P14, P21 and

  the expression and function of efflux transporters in the developing BBB. Although we cannot assume that these observations are responsible for the pharmacoresistance in children with Dravet Syndrome, they suggest nonetheless the importance of brain development on brain permeability and the interaction of VPA, STP and CLB with the developing BBB. The results assembled in this section have been partly presented as an oral communication at the 11 th European Congress on Epileptology in july 2014 in Stockholm, Sweden; as an oral communication at the 19 th Annual Meeting of the French Society of Pharmacology and Therapeutics in april 2015 in Caen, France; and as a poster in 11 th International Conference on Cerebral Vascular Biology in june 2015 in Paris, France. Impact of the Antiepileptic Drugs used in the Dravet Syndrome on the Blood-Brain Barrier in the Immature Rat (To be submitted to "Molecular Pharmaceutics") List of authors: Ricardo Viana Soares 1,2 , Stéphanie Chhun 3 , Vincent Jullien 1 , Tuan Minh Do 2 , Anne-Cécile Guyot 2 , Catherine Chiron 1 , Gérard Pons 1 , Aloïse Mabondzo 2 1 -Inserm U1129, Paris, France; University Paris Descartes; CEA, Gif-sur-Yvette, France 2 -CEA, Direction des Sciences du Vivant, iBiTec-S, Service de Pharmacologie et d'Immunoanalyse, 91191, Gif-sur-Yvette, France 3 -Inserm U1151, INEM, Paris, France; University Paris Descartes. Hôpital Necker-Enfants Malades, 149 rue de Sèvres 75015 Paris, France) Impact of the antiepileptic drugs used in the Dravet syndrome on the blood-brain barrier in the immature rat Authors: Ricardo Viana Soares 1,2 , Stéphanie Chhun 3 , Vincent Jullien 1 , Tuan Minh Do 2 , Anne-Cécile Guyot 2 , Catherine Chiron 1 , Gérard Pons 1 , Aloïse Mabondzo 2* 1 -Inserm U1129, Paris, France; Université Paris Descartes; CEA, Gif-sur-Yvette, France 2 -CEA, Direction des Sciences du Vivant, iBiTec-S, Service de Pharmacologie et d'Immunoanalyse, 91191, Gif-sur-Yvette, France 3 -Inserm U1151, INEM, Paris, France; Université Paris Descartes. Hôpital Necker-Enfants Malades, 149 rue de Sèvres 75015 Paris, France) Key words: Dravet syndrome, immature blood-brain barrier, epilepsy, efflux drug transporters regulation

Figure 1 -Figure 2 -Figure 3 -Figure 4 -Figure 6 -

 12346 Figure 1 -Effects of VPA on the expression of ABC (A) and SLC (B) efflux transporters genes in the microvessels of P14, P21 and P56 rats. Rats (n=5 for P14, n=15 for P21 and P56) were treated with VPA 400 mg/kg/d through osmotic minipumps. Total RNA was analyzed by RT-PCR after extraction from brain microvessels of treated and control rats (* -p<0.05, ** -p<0.01, *** -p<0.001, 2-way-ANOVA, N/Dnot detected).

  Study of the antiepileptic drugs transport through the blood-brain barrier in children (Presented as an oral communication at the 19 th Annual Meeting of the French Society of Pharmacology and Therapeutics in april 2015 in Caen, France) RSoares a,c , S Chhun b , V Jullien a , M Do c , G Noyalet c , C Chiron a , G Pons a , A Mabondzo c A -Inserm U1129 (Infantile Epilepsies and Brain Plasticity), Paris Descartes University, CEA/DSV/I2BM -Paris (France); B -Inserm U1151, INEM, Paris Descartes University, Medicine Faculty Paris Descartes -Paris (France); C -CEA/DSV/iBiTec-S (Service de Pharmacologie et Immunoanalyse) -Paris (France) Introduction: Drug resistant epilepsy remains a major concern in pediatrics despite the large number of anti-epileptic drug (AEDs) available. Resistance to AEDs may be related to adaptive mechanisms that oppose their brain penetration. Recent research has covered the role of the adult Blood-Brain Barrier (BBB) in this protective mechanism through altered expression of efflux pumps e.g. P-glycoprotein (P-gp) and Breast Cancer Resistance protein (BCRP). In the present work we aimed at studying the impact of valproic acid (VPA) on P-gp, BCRP efflux transporters function and in the expression of selected ABC and SLC transporters genes in immature rat brain microvessels. Material and methods: Post-natal day 21 (P21) and P56 rats (n = 5) were treated for 5 days with VPA (400 mg/Kg/days and 350 mg/Kg/days) through subcutaneous osmotic pumps. Changes in BBB transporters P-gP and BCRP function was measured after the infusion of digoxin and prazosin as P-gP

Factors

Figure 5 .

 5 Figure 5. Effects of CBZ on the expression of ABC (A) and SLC (B) efflux transporters genes in the microvessels of P14, P21 and P56 rats. (* -p<0.05, ** -p<0.01, *** -p<0.001, 2-way-ANOVA, N/Dnot detected).

Figure 8 .

 8 Figure 8 . Schematic representation of the in vitro blood-brain barrier model (RBECrat brain endothelial cells)

PFigure 9 .

 9 Figure 9. Interaction of VPA with P-gP (A) and BCRP (B) in an in-vitro model of BBB. Endothelial monolayers were pre-exposed to VPA before adding [ 3 H]VBL or [ 3 H]PRZ. Efflux ratios were calculated as the ratio between the basolateral-to-apical and apical-to-basolateral P app of each substrate. Controls refer to efflux ratios when [VPA]=0 μM.

6. 2 . 4 .

 24 Interaction of VPA with the efflux transporters P-gP and BCRP in an in vitro model of the developing BBB constructed with primary cultures of rat brain endothelial cells

  pharmacoresistance in Childhood Epilepsies. The potential studies that could follow this work should concentrate on the effects of these drugs both independently or in combination in the developing BBB of animal models of epilepsy, in particular the existing model for the Dravet Syndrome. The different impacts of VPA and CLB in the efflux activity of P-gP and BCRP, respectively, should be further studied at a cellular level, for instance in an in vitro model of the developing BBB. Furthermore, the impact of the OAT1 transporter on VPA brain disposition and the identification of the potential mechanisms regarding STP transport through the developing BBB should be developed. The unique characteristics of children with epilepsy, different from the adults, should always be taken seriously into account when studying drugs for Childhood Epilepsies.

  , adapted

	Antiepileptic Drug Abbreviation	Mechanism of action
	Carbamazepine	CBZ	Voltage-gated Na + channel inhibition
	Clobazam	CLB	GABA A receptor agonist
	Eslicarbazepine	ESL	Voltage-gated Na + channel inhibition
	Ethosuximide	ESX	Voltage-gated Ca 2+ channels inhibition
	Felbamate	FLB	NMDA receptor inhibition
	Gabapentin	GBP	Voltage-gated Ca 2+ channels inhibition
	Lamotrigine	LTG	Voltage-gated Ca 2+ and Na + channels inhibition
	Levetiracetam	LEV	SV 2 A protein inhibitor
	Oxcarbazepine	OXC	Voltage-gated Na + channel inhibition
	Perampanel	PER	AMPA receptor inhibition
	Phenobarbital	PB	GABA A receptor agonist
	Phenytoin	PHT	Voltage-gated Na + channel inhibition
	Stiripentol	STP	GABA A receptor agonist
	Tiagabine	TGB	GABA transporter inhibition
	Topiramate	TPM	Voltage-gated Na + channel inhibition and increased GABA levels
	Valproic acid	VPA	Voltage-gated Na + channel inhibition and increased GABA levels
	Vigabatrin	VGB	GABA transporter inhibition
	Zonisamide	ZNS	Voltage-gated Na + channel inhibition

  . In refractory cases, surgery is an option to consider in focal epilepsy, at any age including the youngest ones.Except the Benign epilepsy with centro-temporal spikes previously mentioned, which is probably genetically determined and vanishes before adulthood in all cases, pure focal epilepsies are due to a cortical brain lesion. Lesion may be acquired (tumor, cerebral traumatism, cerebral infarct, and hippocampal sclerosis) or developmental and therefore present before birth (malformation, anoxoischemia, and antenatal infection). By contrast with adults, developmental lesions are the most frequent cause of pediatric focal epilepsies, mostly malformations of cortical development, with Focal

  11

  11

Table 4 .

 4 Comparison between human and rat brain development.

	Rat Human	Birth Start of 3 rd trimester	3 rd trimester	P10-12 Birth/ Newborn	Infants	P21 2 nd year	Children	P28 10-11 years	Adolescents	P42 18years

  Transwell® inserts co-cultured with glial cells were validated with [ 14 C]sucrose A-to-B apparent permeability (integrity marker) and [ 3 H]vinblastine efflux ratio (P-gP substrate). Briefly, separate 0.1 μCi/mL of [ 14 C]sucrose and [ 3 H]vinblastine solutions (Perkin Elmer) were prepared in warm transfer buffer (150 mM NaCl, 5.2 mM KCl, 2.2 mM CaCl 2 , 0.2 mM MgCl 2 , 6 mM NaHCO 3 , 2.8 mM glucose, and 5 mM HEPES) and loaded in the donor compartment (apical for A-to-B transport using [ 14 C]sucrose and [ 3 H]vinblastine, basolateral for B-to-A transport only using [ 3 H]vinblastine). Receptor compartment was loaded with warm transport buffer. After one hour incubation at 37ºC under agitation, samples were drawn from all compartments, mixed with scintillation counting mix (Biofluor Plus, Perkin Elmer) and disintegrations counted in a Perkin Elmer

LKB Rackbeta (Perkin Elmer). Co-culture batches with [ 14 C]sucrose apparent permeability below 8x10 -6 cm/s and 3 H-vinblastine efflux ratio above 2 were considered valid for transport studies.

To study the VPA impact on in vitro P-gP and BCRP efflux activity, the endothelial cells monolayer in Transwell® inserts co-cultured with glial cells from both adult and immature rats were transferred to new 12-well plates and exposed to VPA at 60, 20, 2, 0.2 and 0.02 mg/L in warm transfer buffer.

After 15 min, [ 3 H]vinblastine (VBL, P-gP substrate) and [ 3 H]prazosin (PRZ, BCRP substrate) were added to the apical compartment (A-to-B transport, AB) or basolateral (B-to-A transport, BA) for a final concentration of 0.1 μCi/mL. After one hour incubation at 37ºC under agitation, samples were drawn from all compartments, mixed with scintillation counting mix (Biofluor Plus, Perkin Elmer) and disintegrations counted in a Perkin Elmer LKB Rackbeta (Perkin Elmer).

Table 1 -

 1 List of in vitro BBB models constructed from primary cultures of brain endothelial cells

	Article	Origin of	Age	Mono/	Age of AC Functional	Morphologic	Drug screening	Biblio
		EC	of	Co-		Characteri	al		graph
			EC	culture		zation	Characteriza		y
							tion of EC
	Perrière et al	Rat	P14	Co	AC P1	TEER;	ZO-1,	Doxorubicin,	32
	(2007)					Sucrose	claudin-3,	diazepam,
						permeabilit	claudin-5,	morphine-6-
						y;	occludin,	glucoronide,
						rhodamine	beta-catenin,	inulin,
						123 as P-	PECAM-1,	imipramine,
						gP	Oatp2, Bcrp	prazosin,
						substrate	and P-gP	colchicine,
						and		vincristine,
						daunorubic		vinblastine,
						ine as		glucose,
						BCRP and		fluorescein,
						MRP		FITC-labeled
						substrate		dextrans.
	Mabondzo et	Human	Adult Co	Adult AC	Sucrose	ZO-1,	Vinblastin,	77
	al. (2010)					permeabilit	claudin-3,	digoxin,
						y	claudin-5,	haloperidol,
							SLC and	propranolol,
							ABC	etoposide,
							transporters	taurocholic acid,
								fluorodesoxyglu
								cose, fluoro-
								Dopa, 2-fluoro-
								A-85380,
								raclopride,
								flumazenil,
								befloxatone, PE-
								2I,
								glucuronideaceta
								minophen 1-
								hydroxymidazol
								am, rosuvastatin,
								acetaminophen,
								midazolam,
								rosuvastatin,
								dextrometorphan
								e and caffeine.
	Persidsky et	Human	Adult	Co	12-20wk of	N/A	vWF	N/A
	al. (1997)		, and		gestation
			3-7
			years
			old

  -ΔCt , where ΔCt refers to difference between the threshold cycle (Ct) of the target gene and the Cyclophylin A gene (Ppia) as the control housekeeping gene (ΔCt = Ct target -Ct Ppia ). Cyclophylin A itself has a stable expression throughout the 3 age classes with a mean Ct of 18.3 (CV% = 8%). PCR threshold cycles over 35 were considered non-reliable and discarded. Specificity of the PCR primers was controlled by melting curve

	analysis.
	P-gP and BCRP functionality assessment: P14, P21 and P56 rats were exposed to a cocktail

containing digoxin (DGX, 12mg/kg/d), prazosin (PRZ) and atenolol (ATN, both at 6mg/kg/d) dissolved in DMSO:PEG 200 (all substrates from Sigma-Aldrich, Saint-Quentin Fallavier, France) through a subcutaneously implanted osmotic pump (Alzet ® model 2001D, 8 ìL/h). After a 4h

Table I -

 I Selected ABC and SLC genes and their antiepileptic drugs substrates (PHT -phenytoin, PB -phenobarbital, OXC -oxcarbazepine, ESL -eslicarbazepine, LEV -levetiracetam, LTGlamotrigine, CLB -clobazam, VPA -valproic acid).

	Protein	Accession # /Reference	Gene	AEDs
	P-gP	NM_133401

  -ΔΔCt , where ΔΔCt refers to difference between the threshold cycle of the target gene and Cyclophilin A (Ppia) as the control housekeeping gene in treated and control animals (ΔΔCt = ΔCt treated -ΔCt control , ΔCt= Ct target -Ct Ppia ). Specificity of the PCR reactions was analyzed by melting curve analysis. P21 and P56 rats were treated for five days with 400 mg/kg/d VPA, 250 mg/kg/d STP and 4 mg/kg/d CLB through subcutaneous osmotic pumps, plasma and brain tissue were collected as described for P-gP and BCRP functionality and storeded at -20ºC until dosage. Brains were first homogenized with water (2 mL per gram of tissue) in an UltraTurax. For VPA dosage, brain homogenates were first centrifuged at 21000g for 10 min and 100 μL of the supernatant and of plasma were treated with 300 μL of methanol and centrifuged for 10 min at 13000g. Methanolic extracts were evaporated under N 2 current and dissolved in an ammonium acetate 10 mM : methanol (1:1) mix. For CLB and STP, 100 μL of plasma and 500 μL of brain homogenate were treated with 2 mL of tertbutylmethylether and 3 mL of n-pentane respectively. Samples were agitated for 15 min, frozen at -20ºC, the supernatant evaporated under N 2 current and the dry STP residues dissolved in ammonium acetate 10 mM : methanol (1:1), while CLB were dissolved in acetic acid 0.1% : methanol (1:1).Analysis of the AEDs was made by LC-MS in a TSQ Vantage coupled to an UPLC Ultimate 3000 (Thermofisher, Villebon-sur-Yvette France) using a Waters BEH C18 column (5 x 2.1 mm, 1.7 µm).Experiments were planned with a randomized complete block design for two factors (Age, 2 degrees of freedom; and Treatment, 1 degree of freedom). Data from P-gP and BCRP efflux activity (K p,brain/plasma ) and gene expression analysis (Ct) were analyzed by the two-way analysis of variance test (2-way ANOVA), followed by the Student-Bonferroni post-test. Whenever conditions for 2-way ANOVA were not met (normality and homoscedasticity), t-Student test or Mann-Whitney test were performed to compare treated and control rats in each class of age. The presented 2 -Ct standard deviations are derived from propagation of uncertainty of the Ct of both controls and treated rats.STP and CBL K p,brain/plasma between P14, P21 and P56 rats were analyzed by one-way analysis of variance, followed by Student-Bonferroni post-test. VPA data was analyzed by the Kruskal-Wallis non parametric test for analysis of variance by ranks. Results were considered statistically significant if p<0.05. Data was analyzed with Prism 5.0 and Excel software and data are presented as mean ± SD.

	DGX, PRZ and ATN dosage by LC/MS DGX, PRZ and ATN concentrations were measured in brain homogenates and plasma by LC/MS-MS, using digoxin-d3 and atenolol-d7 as internal standards. Brains were first homogenized with water (2mL per g of brain tissue) in an Ultra-Turax. Brain homogenates (400μL) were separately treated with 1 mL methanol for protein precipitation, centrifuged at 20000g for 15 min at 4 °C, the supernatant collected and evaporated at 40°C under N 2 current. Both brain homogenates residues and plasma (150μL) samples were passed through Isolute® columns: samples were mixed with ammonia 0.75 M : methanol (4:1), loaded on to the columns and eluted with 2 mL of dichloromethane : isopropanol (7:3) followed by 2 mL dichloromethane : isopropanol (7:3) + 0.2% formic acid. After evaporation at 40ºC under N 2 current, samples were dissolved in 200μL of mobile phase (ammonium acetate 5mM : methanol, 95:5). LC/MS-MS analysis was performed in a Nexera X2 UHPLC system (Shimadzu, Noisiel, France) coupled to a triple quadrupole Finnigan TSQ Quantum Ultra mass P14, Mobile phase A consisted of ammonium acetate 5% and phase B of pure methanol. For VPA and STP,
	spectrometer (Thermo Scientific, Villebon-sur-Yvette France), using a Phenomenex RP C18 HPLC runs started with 50% phase B for 1 min that increased up to 90% until 3 min. For CLB, runs started
	column (3 x 100 mm, 2.6 μm). Mobile phase A consisted in ammonium acetate 5 mM : methanol (1:1) with 2% phase B that linearly increased to 70% until 7 min. VPA was monitored in a negative
	and phase B pure methanol. Runs started with 95% phase A for 1 min that decreased steadily to 5% electrospray ionization mode while STP and CLB in positive mode (3 kV spray voltage, gas
	for 3 min, followed for 1 min with 5% phase A. Analytes were monitored in a positive electrospray temperature 290 ºC). VPA was measured in simple MS mode (m/z 143), while for STP m/z 217.1>159
	ionization mode (3 kV spray voltage, gas temperature 200 ºC), following transition m/z 798.5 > 651.4 and for CLB m/z 301>259.07 transitions were followed. Internal standards were used throughout the
	for digoxine, m/z 384.2 > 247.1 for prazosine and m/z 267.2 > 145.1 for atenolol. Data were collected experiments: VPA-D6, STP-D9 and nCLB-D6 for VPA, STP and CLB respectively. Data were
	with Xcalibur software (vs. 2.1, Thermo Scientific, Villebon-sur-Yvette France) and analyzed with collected with Xcalibur software (vs. 2.1, Thermo Scientific, Villebon-sur-Yvette France) and
	LCQuan software (vs. 6.1, Thermo Scientific, Villebon-sur-Yvette France). analyzed with LCQuan software (vs. 6.1, Thermo Scientific, Villebon-sur-Yvette France). Brain/blood
	AEDs in vivo brain/plasma partition coefficients partition coefficients were calculated by the following equation, where brain concentrations were
	corrected by subtracting blood contamination that was considered as 2%:
	P-gP and BCRP functionality and the impact of the AED treatments on the efflux transporter
	functionality were assessed by calculation of the K p,brain/plasma of DGX and PRZ.
	Statistical analysis

Table 1 -

 1 Selected ABC and SLC genes.

	Protein	Accession # /Reference	Gene
	P-gp	NM_133401	Abcb1a
	P-gp	NM_012623	Abcb1b
	Mrp1	NM_022281.2	Abcc1
	Mrp2	NM_012833.2	Abcc2
	Mrp4	NM_133411	Abcc4
	Mrp5	NM_053924	Abcc5
	Mrp6	NM_031013.1	Abcc6
	Bcrp	NM_181381	Abcg2
	Eaat3	NM_013032.3	Slc1a1
	Oat1	NM_017224.2	Slc22a6
	Oat3	NM_031332.1	Slc22a8
	Mct1	NM_012716	Slc16a1
	Cyclophilin A	NM_017101.1	Ppia

Table 2 -

 2 ΔCt values (ΔCt = Ct targetgene -ΔCt housekeepinggene ) of control and treated rats were compared by two-way-ANOVA, followed by Bonferroni post-hoc test to identify differences between treated and control groups within each age class.

			Legend: A -Age; T -Treatment; I -Interaction; * -p<0.05; ** -
	p< 0.01; *** -p<0.001, N/T -not tested.
		VPA		STP		CLB
	Genes
		Factors	F values	Factors	F values	Factors	F values
	Abcb1a (P-gp)	I*	4,95	N/T		I*	3,844
	Abcb1b (P-gp)			A*	4,403	I*	5,614
	Abcg2 (Bcrp)	N/T		A**	7,030	T*	7,070
	Abcc1 (Mrp1)	A**, T*	10,31 (A), 4,698 (T),	I***	28,86	I***	65,44
	Abcc2
	(Mrp2)
	Abcc4 (Mrp4)					A***	14,01
	Abcc5 (Mrp5)			T*	4,634	I***	21,43
	Abcc6
	(Mrp6)
	Slc1a1 (Eaat3)			A***	11,38	N/T
	Slc16a1 (Mct1)	N/T		A***, T**	58,00 (A), 8,709 (T)	I**	5,759
	Slc22a6 (Oat1)	I**	9,163	N/T		N/T
	Slc22a8 (Oat3)			I***	13,00	I***	36,66

Table 3 -

 3 Impact of VPA, STP and CLB treatment on the efflux activity of P-gP and BCRP efflux transporters in the BBB at post-natal days14, 21 and 56 (P14, P21 and P56 respectively). DGX and PRZ K p,brain/plasma of control and treated rats were compared by two-way-ANOVA, followed by Bonferroni post-hoc test to identify differences between treated and control groups within each age class. Legend: A -Age; T -Treatment; I -Interaction; * -p<0.05; ** -p< 0.01; *** -p<0.001, N/Tnot tested. To study the impact of the anti-epileptic drugs (AEDs) VPA, STP, CLB and CBZ, on P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) efflux transporters function in immature rats. Drug resistant epilepsy remains a major concern in pediatrics despite the different AEDs available. Resistance to AEDs may be related to adaptive mechanisms that oppose their penetration into the brain. Recent research has covered the role of the adult Blood-Brain Barrier (BBB) in this protective mechanism through altered expression of efflux pumps e.g. P-gP and BCRP. In this work we aim to assess modifications of those efflux pumps function that could originate AEDs resistance in immature animals. We will focus on the AEDs used in the Dravet syndrome

		VPA		STP		CLB
	Efflux
	transporter	Factors	F values	Factors	F values	Factors	F values
	P-gp	I**	8,122	A***	87,22	A***	103,5
	Bcrp			A***	54,58	I***	28,69

Table 1 -

 1 Study of the Antiepileptic Drugs Impact in the Developing Rat Blood-Brain Barrier (Presented as a poster in 11 th International Conference on Cerebral Vascular Biology in june 2015 in Our results show the impact of VPA, STP, CLB and CBZ treatment on the BBB transporters phenotype. Moreover, all studied AEDs induced gene expression levels modifications that were unique to certain age classes. P-gp function was also found to decrease significantly after treatment with VPA in both P21 and P56 rats. The ongoing research will unravel the effects of STP, CLB and CBZ on the P-gp and Bcrp function and all data will be presented. Effects of valproic acid (VPA), stiripentol (STP), clobazam (CLB) and carbamazepine (CBZ) on the expression of the selected ABC and SLC transporters. A -Age; T -Treatment; I -Interaction;  -Increase in expression;  -Decrease in expression; * -p<0.05; ** -p<0.01; *** -p<0.001

	Paris, France)
	Ricardo V. Soares 1,2 , Stéphanie Chhun 3 , Tuan Minh Do 2 , Gaël Noyalet 2 , Vincent Jullien 1 , Catherine
	Chiron 1 , Gérard Pons 1 , Aloïse Mabondzo 2
	1 INSERM U1129 "Infantile Epilepsies and Brain Plasticity", Paris, France; Université Paris Descartes,
	Sorbonne Paris Cité; CEA, Gif-sur-Yvette, France
	2 CEA/DSV/iBiTec-S -Pharmacology and Immunoanalysis Department, Gif-sur-Yvette, France
	3 Inserm U1151, INEM, Paris Descartes University, Medicine Faculty Paris Descartes, Paris, France
	Methods: Post-natal day 21 (P21) and P56 rats (n=5 per group) were treated for 5 days either with
	VPA (400mg/Kg/d and 350mg/Kg/d), CBZ (50 mg/Kg/d), STP (250 mg/Kg/d) or CLB (4 mg/Kg/d)
	through subcutaneous osmotic pumps. P-gp and Bcrp function was measured after the infusion of
	specific substrates (digoxin and prazosin, respectively) by calculation of brain/blood partition
	coefficients (Kp,brain) by measuring blood and brain concentrations with LC-MS in steady-state
	conditions. The same AEDs treatment was applied in parallel groups (n=5) of P14, P21 and P56 rats
	for qPCR analysis of selected ABC and SLC genes expression levels in brain microvessels. Data were
	analyzed by 2-way ANOVA followed by Bonferroni post-hoc tests and considered significant if
	p≤0.05.
	Results: Changes in ABC and SLC transporters expression were linked to age and treatment, both
	factors acting independently, but also interactions were observed (Table 1). STP effects on ABC and
	SLC genes expression were exclusively observed on P14 rats. VPA decreased significantly Abcb1a
	gene expression in P14 rats (p<0.05), while all other altered genes increased their expression. After 5
	days treatment with VPA, digoxin Kp increased from 0.070±0.01 to 0.136±0.016 in P21 rats and from
	0.042±0.02 to 0.077±0.07 in P56, both differences being statistically significant (p<0.01). A trend to

Purpose: Recent research has covered the role of the adult Blood-Brain Barrier (BBB) as a protective mechanism by differential expression of xenobiotic-efflux related ATP Binding Cassette (ABC) and Solute Carrier (SLC) transporters. In this work we aim to assess modifications of ABC and SLC transporters that could originate resistance to the selected anti-epileptic drugs (AEDs) valproic acid (VPA), stiripentol (STP), clobazam (CLB) and carbamazepine (CBZ) in developing animals with specific focus on P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (Bcrp) efflux transporters function.

  The Abcc5 gene was independently influenced by both age and treatment. When comparing treated with controls, CBZ only induced genes of the ABC family (standard deviation) (figure): Abcb1b was induced 6,0-fold (0,28) in P14 rats, Abcc1 by 2,1-fold (0,22) in P56 and Abcc5 by 1,6 (0,25) also in P56 rats. In parallel with VPA, STP and CLB, Abcc2 gene was either undetected or originated Ct values over 35 in all groups.

Table 5 .

 5 Impact of CBZ treatment on the mRNA levels of selected ABC and SLC transporters in isolated rat

			CBZ
	Genes
		Factors	F values
	Abcb1a (P-gp)	A***	28,95
	Abcb1b (P-gp)	I***	7,54
	Abcg2 (Bcrp)	N/T
	Abcc1 (Mrp1)	I***	11,62
	Abcc2 (Mrp2)
	Abcc4 (Mrp4)	A***	26,11
	Abcc5 (Mrp5)	A***, T* 13,81 (A), 4,75 (T)
	Abcc6 (Mrp6)	N/T
	Slc1a1 (Eaat3)	N/T
	Slc16a1 (Mct1)	I*, A*	4,19 (I), 20,85(A)
	Slc22a6 (Oat1)	A***	26,33
	Slc22a8 (Oat3)	A***	26,46

brain microvessels at post-natal days 14, 21 and 56. Legend: A -Age; T -Treatment; I -Interaction; * -p<0.05; ** -p< 0.01; *** -p<0.001, N/Tnot tested.

Table 6 .

 6 Impact of the CBZ treatment on the efflux activity of P-gP and BCRP efflux transporters in the BBB at post-natal days 14, 21 and 56 (P14, P21 and P56 respectively). DGX and PRZ K p,brain/plasma of control and treated rats were compared by two-way-ANOVA, followed by Bonferroni post-hoc test to identify differences between treated and control groups within each age class.

	Legend: A -Age; T -Treatment; I -Interaction; * -p<0.05; ** -

(Place here table I)

References

Acknowledgements

I would like to thank my thesis supervisors Pr. Gérard Pons and Dr. Aloïse Mabondzo for having accepted me as their PhD student and their roles as supervisors during this work. This is a once in a life opportunity. I am deeply grateful for allowing me to work with your research teams and for the very interesting subject of this work I would like to thank the Agence National de la Recherche for the finantial support through the Neuratris project. I would like to thank Dr. Catherine Chiron for the opportunity to work in the Inserm unity UMR 1129, for her precious help in the revision of this work and articles, and also for the finantial support of the UMR 1129 I would like to thank Dr. Christophe Junot and Dr. Christophe Creminon for the opportunity to work at the Pharmacology and Immunoanalysis Service with a very good team. I would like to thank Pr. Robert Farinotti and Dr. Chantal Barin-Le Guellec for accepting to be revisors of this work and make part of the jury. Also, I would like to thank Dr. Astrid Nehlig and Dr. Pascal Clayette for accepting to participate as jury members during the PhD dissertation. I would like to thank Dr. Stéphanie Chhun and Dr. Vincent Jullien for their continous help and advising throughout the experiments. Their comments and revision of the articles are also very important. I would like to thank my closer partners at Pharmacology and Immunoanalysis Service for their support and help during this work: Charlotte Leuxe, Clemence Disdier, Anne-Cécile Guyot, Emilie Jaumain, Tuan Minh Do, Jerôme Telliet, Gael Noyalet and Gabriella Ullio. This work is dedicated in first place to my family, my mother and my sister who helped and continuously supported me throughout the days of work in France. I am deeply thankfull for their support.

This work is also dedicated to my closest friends, especially Ana Luísa Amorim for our long friendship and the continous support throughout the years. The friends I have met in France are also thanked: Carina Silva, João Pedro Cabral, Anabela Costa, Jorge Corsino and Ademar Pozzatti Junior.

Acknowledgments

The authors would like to thank Catherine Chiron and Vincent Jullien (Inserm U1129) for their scientific support and the French Agence Nationale de Recherche for the financial support (NeurATRIS initiative).

Abstract

The Blood-Brain Barrier (BBB) is responsible for the control of solutes' concentration in the brain. Tight junctions and multiple ATP-binding cassette (ABC) and SoLute Carrier (SLC) efflux transporters protect brain cells from xenobiotics, therefore reducing brain exposure to intentionally

Abstract

The Blood-Brain Barrier (BBB) is the most important interface between brain parenchyma and blood.

It presents complex tight junction and multiple transporters belonging to the ATP-binding cassette and Solute Carrier families in the surface of the brain endothelial cells. The intricate mechanisms through which brain cells modulate the formation of the BBB during brain development may have an impact on the pattern of surface transporters. In vitro BBB models have been developed as alternatives to study transport of substances across the BBB. We have analyzed the published BBB models constructed with primary cultures of brain endothelial cells in order to examine if age-related differences were taken in to account to perform permeability studies. We observed that the majority of the published BBB models did not mention the age of the animals from where the endothelial cells were isolated. Only one published BBB model has analyzed differences in permeability between the adult and immature brain. We conclude that the exploitation of in vitro BBB models for the screening of potential drugs for the pediatric population should include age-related differences in the pattern of transporters of the developing BBB.

Abstract

Dravet Syndrome (DS) is a severe infantile onset epilepsy syndrome. Despite of a significant efficacy of the currently recommended tritherapy -stiripentol, valproic acid, clobazampatients remain with recurrent seizures. Blood-Brain Barrier (BBB) changes in permeability, which could limit antiepileptic drug (AEDs) transport by efflux transporters has been considered at least in part as responsible for pharmacoresistance in epilepsy. We explored the potential impact of immaturity related to age and of the AED treatment itself on the permeability of the BBB to the AED tritherapy used in the Dravet syndrome patients in developing rats. We investigated the impact of each of the three AEDs on the expression pattern of ABC and SLC families' efflux transporters as well as on P-glycoprotein (P-gP) and Breast Cancer Resistance Protein (BCRP) efflux activity. Valproic acid, stiripentol and clobazam brain permeability was different between immature (post-natal day (P) 14 and 21) and adult rats (P56).

The three AEDs studied modified the expression of the BBB transporters genes. Stiripentol increased P14 rat expression of ABC and SLC genes. Clobazam showed inhibition of Abcc1, Abcc5, Slc1a1, Slc16a1, Slc22a6 and Slc22a8 in P14 rats, but induction in P21 or P56 rats. Valproic acid seemed to influence P-gP in P14 and in P21 and clobazam seemed to decrease BCRP efflux activity only in P21.

These results suggested that the interaction between the developing BBB and the AEDs could be different in immature BBB compared to adult animals.

Material and Methods

Animals

Post-natal days 14, 21 and 56 rats (P14, P21 and P56, respectively) were purchased from Janvier (Saint-Berthevin, France). They were housed at least for 2 days before the experiments in a 12h light dark-cycle with standard food (LASQC diet Rod16-H, LASvendi, Germany) and water ad libitum. All

Chemicals

Clobazam (CLB), valproic acid (VPA), digoxin (DGX), atenolol (ATN) and prazosin (PRZ) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Stiripentol (STP) was a kind gift from Biocodex (Gentilly, France). Dimethylsulphoxide (DMSO), propylene glycol (PPG), polyethylene glycol 200 (PEG200) and ethanol were also obtained from Sigma-Aldrich.

day treatment with the AEDs

Rats were exposed subcutaneously to VPA, STP or CLB independently through osmotic mini pumps (Alzet ® model 2001, 1 μL/h Saint-Germain-Nuelles, France). VPA was dissolved in saline, the remaining drugs dissolved in DMSO : propylene glycol : ethanol (45:45:1). Rats were treated with 400mg/kg/day (P21) and 350mg/kg/d (P56) for VPA, 250mg/kg/d for STP and 4mg/kg/d for CLB. These doses were selected previously by a dose-ranging study in order to achieve the plasma concentrations in rats that are considered therapeutic in children (data not shown). Pumps were prepared the day before and let in saline overnight at 37ºC for priming. For osmotic pump implantation, rats were first anesthetized with isoflurane (3-4% v/v, 1.5mL/min) in an induction chamber and maintained under continuous isoflurane anesthesia through a mask. Rats were shaved on

Results

Impact of VPA, STP and CLB on ABC and SLC genes expression

The results obtained showed an impact of VPA, STP and CLB treatment on mRNA levels of selected ABC and SLC transporters in isolated rat brain microvessels at post-natal days 14, 21 and 56 (P14, P21 and P56 respectively). Also significant interactions with age were observed with the three AEDs:

VPA treatment interacted with age on Abcb1a and Slc22a6 gene, STP on Abcc1 and Slc22a8, CLB on Abcb1a, Abcb1b, Abcc1, Abcc5, Slc16a1, and Slc22a8 (Table 2).

Considering VPA (Figure 1), the effects were limited to immature rats: a 33% decrease (SD = 0,195) in mRNA Abcb1a gene levels in P14 rats and a 4,1 fold (SD = 0,49) increase in Slc22a6 gene expression in P21. STP effects on the expression of ABC and SLC transporters were limited to P14 rats and exclusively induced the expression of genes (Figure 2): Abcc1 gene was induced 3.7-fold (SD = 0,16), Abcc5 1.5-fold (SD = 0.14), Slc16a1 1.9-fold (SD = 0.16) and Slc22a8 6.1-fold (SD = 0,20).

CLB showed both induction and inhibition of the genes expression in all classes of age (Figure 3).

Regarding the ABC transporters, CLB induced Abcb1a gene expression in P14 rats by 1.7-fold (SD = 0,21); regarding the Abcc1 gene, its expression was decreased in P14 rats by 65% (SD = 0.16) of controls, however it was increased by 2.3-fold (SD = 0.22) in P21 and 2.3-fold (SD = 0.24) in P56; with respect to Abcc5, its expression was inhibited by 48% (SD = 0.23) in P14 rats and induced 1.7 fold (SD = 0.34) in P56 rats (Figure 3A). The studied SLC genes were all inhibited in P14 rats:

Slc1a1decreased by 75% (SD = 0.31), Slc16a1 by 50% (SD = 0.22), Slc22a6 by 65% (SD = 0.34), and Slc22a8 by 90% (SD = 0.30). Slc1a1 was however induced in P21 rats by 3.0-fold (SD = 0,24) and in P56 by 2.8 (SD = 0,36). Slc22a6 was also induced in P21 by 7.0-fold (SD = 0,34) (Figure 3 B)

Impact of VPA, STP and CLB on P-gP and BCRP functionality

The results obtained showed an impact of VPA, STP and CLB treatment on P-gP and BCRP functionality assessed by calculation of the K p,brain/plasma of DGX and PRZ, respectively, in rats treated for 5 days with each AED under study and compared with controls (vehicle). The 2-way ANOVA Treatment with VPA seemed to influence the expression of various genes in the BBB. VPA reduced the expression of Abcb1a in P14 rats. However VPA was shown not to be a substrate of P-gP and therefore this effect should not impact VPA distribution in the brain but may be responsible for interactions with other P-gP substrates 24,25 . On the other hand, VPA induced the expression of Slc22a6 in P14 and P21: this is interesting because VPA is an Oat1 substrate, being secreted also in the kidney proximal convoluted tubule 26,27 . Furthermore, Oat1 and Oat3 are present in the choroid plexus, the brain structure corresponding to the blood-cerebrospinal fluid barrier 28 . It is difficult to determine whether this transporter has a considerable influence on VPA disposition in the brain: apparently this transporter is expressed in the brain but information about its actual role in the BBB (either influx or efflux from the brain) is lacking 29 . Moreover, VPA influx and efflux has also been studied in rabbit microvessels, and an active transport sensitive to inhibition by p-aminohippurate was detected 30,31 . In addition VPA has been shown to be transported by Mct1 (Slc16a1), which is highly expressed in the immature brain to supply energy substrates 32-34 .

An apparent discrepancy between the effects of VPA on the expression of Abcb1a and the in vivo P-gP efflux activity was observed: a decrease in Abcb1a gene expression and an apparent increase in P-gPmediated DGX efflux. This may be related to post-transcriptional modulation by VPA of the Abcb1a gene product, or at molecular level such as an interaction between VPA and the P-gP protein.

The results related to STP administration showed an apparent increase in its distribution to brain with increasing age that may be due to BBB maturation: STP K p,brain/plasma steadily increased from P14 to P56 after 5 days of treatment. The observed K p,brain/plasma values in the present study were similar to the values already reported for adult rats 35 . The K p,brain/plasma was compared between P21 and P75 rats and no difference was detected 36 . However these results were obtained after one single injection of STP and brain and plasma concentration were not measured at steady-state. If our results on STP K p,brain/plasma in P14 and P21 rats are taken in account, possibly the higher antiepileptic effect of STP in the immature rat reported in the mentioned study 36 may be reduced whenever chronic STP is administered. STP only affected the expression of the selected ABC and SLC genes in P14 rats,