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Rezumatul tezei de doctorat

IMPLICAIILE POLIMORFISMELOR GENELOR MDR1, CYP 2C9 I CYP 2C19 N
EPILEPSIA REFRACTAR

Doctorand: Octavia Sabin
Conductor: Prof. Dr. Anca-Dana Buzoianu


Cuvinte cheie: epilepsie refractar, proteina de eflux, enzime de metabolizare, polimorfism
genetic, MDR1, CYP2C9, CYP2C19

CUPRINS

INTRODUCERE 13
STADIUL ACTUAL AL CUNOATERII
1. Epilepsia definiie, clasificare 17
2. Farmacogenetica epilepsiei 19
2.1. Ipoteze privind cauzele epilepsiei refractare 20
2.2. Variabilitatea sistemului CYP 450 21
2.3 Glicoproteina P
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CONTRIBUIA PERSONAL
1. Ipoteza de lucru/obiective generale 39
2. Metodologie general 39
3. Studiul 1 Evaluarea frecvenei polimorfismelor genelor
CYP2C9, CYP2C19 i MDR1 la epileptici
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4. Studiul 2 Corelarea concentraiilor de valproat cu
polimorfismele CYP2C9, CYP2C19 i MDR1
69
5. Studiul 3 Studiul polimorfismelor genei MDR1 n epilepsia
refractar
101
6. Concluzii generale (sintez) 119
7. Originalitatea i contribuiile inovative ale tezei 121
REFERINE 123
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STADIUL ACTUAL AL CUNOATERII

Farmacogenomica cerceteaz modul n care variaiile genomului uman influeneaz
rspunsul la medicamente i permite adaptarea medicaiei la constelaia genetic individual a
pacientului sub forma tratamentelor individualizate, croite pe bolnav, cu eficacitate i siguran
crescute. Primii pai au fost fcui prin studierea unor mutaii specifice n anumite tipuri de
cancer, ceea ce a permis utilizarea unor medicamente anticanceroase specifice. Astfel, analiza
genotipic naintea administrrii medicamentelor este absolut necesar n anumite situaii
particulare, dar pare s fie o abordare clinic promitoare i n tratamentul altor maladii, n
vederea reducerii efectelor adverse ale medicamentelor i creterea eficacitii acestora.
Epilepsia reprezint un grup de boli neurologice cronice caracterizate prin manifestri
paroxistice recurente prezente la peste 50 de milioane de persoane n lume (inciden de 0.5-1%),
caracterizate prin episoade recurente critice convulsive sau non-convulsive, provocate de
descrcri focale sau generalizate cerebrale. Acestea pot fi controlate medicamentos la peste
dou treimi din pacieni, dar rmne un numr foarte mare de persoane la care crizele epileptice
sunt dificil de controlat, n ciuda unui tratament antiepileptic bine condus i adecvat. Formele
refractare la tratament sunt asociate cu un risc crescut de mortalitate i consecine psihosociale
debilitante.
Una din cele mai la ndemn ipoteze este modificarea farmacocineticii medicamentelor
antiepileptice, de exemplu variabilitatea afinitii enzimelor de metabolizare. Mutaiile n genele
CYP pot produce abolirea, reducerea, alterarea sau creterea activitii enzimatice, respectiv mai
multe exprimri fenotipice ale indivizilor: metabolizatori leni, intermediari, rapizi i
metabolizatori ultrarapizi. Prin obinerea unor concentraii plasmatice subterapeutice, rspunsul
terapeutic poate s fie nesatisfctor.
Pentru pacienii la care se ating concentraii plasmatice corespunztoare, au fost emise mai
multe ipoteze privind etiologia multirezistenei la tratamentul antiepileptic. Principalele dou
concepte care ncearc s explice aceast multirezisten sunt 1) ipoteza modificrii intei de
aciune i 2) ipoteza modificrii funciei proteinelor de eflux. Prima ipotez se bazeaz pe studii
care au artat c unele modificri structurale sau anomalii la nivelul reelei neuronale i n
funcionarea unor neurotransmitori care apar la pacienii epileptici, pot fi cauze de rezisten
la tratamentul farmacologic antiepileptic, adic boala este caracterizat de modificarea intei de
aciune. A doua ipotez a pornit de la observaia c unul din principalele obstacole n tratarea
bolilor cerebrale, inclusiv epilepsia, este slaba penetrare prin bariera fiziologic ce separ esutul
cerebral de snge, bariera hemato-encefalic (BHE). Pe lng jonciunile intercelulare foarte
strnse, la nivelul BHE exist o densitate crescut a transportorilor ABC care confer rezisten
multipl medicamentoas. Pornind de la aceast idee i de la observaia c pacienii cu epilepsie
refractar la tratament sunt nonresponsivi la mai multe antiepileptice, care acioneaz prin
diferite mecanisme moleculare de aciune clinice, s-a sugerat intervenia unui mecanism
nespecific care limiteaz eficacitatea antiepilepticelor, prin supraexprimrea transportorilor de
eflux la nivelul BHE. Prototipul acestor transportori este glicoproteina P (P-gp).
Enzimele citocromului P450 prezente n ficat i tractul gastro-intestinal au o istorie
ndelungat de studii farmacogenetice, care au dovedit o relevan clinic a polimorfismelor, n
special a CYP2C9, CYP2C19 i CYP2D6. CYP2C9 este implicat n metabolismul a mai mult
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de 100 de medicamente, incluznd anticoagulantele orale cumarinice, sulfonilureele, inhibitorii
de angiotensin i o parte din antiinflamatoarele nonsteroidiene. Studiile n vitro au artat c
CYP2C9*3 este asociat cu scderea cleareance-ului intrinsec al medicamentelor ntr-o mai mare
msur dect CYP2C9*2. Cel puin una din variantele CYP2C9*2 sau*3 sunt ntlnite la
aproximativ 20% i respectiv 12 % din populaia caucazian, aproximativ 2,5 % sunt homozigoi
pentru aceste alele sau heterozigoi tip *2/*3 . Rmn dou treimi din populaia caucazian cu
tipul slbatic, cu activitate enzimatic normal. Pentru CYP2C19, homozigoii pentru alelele de
tip slbatic la nivelul exonului 5 i 4 (1*/1*) au fost clasificai ca metabolizatori rapizi
homozigoi (homMR), mutaiile de tip *1/*2, *1/*3 ca metabolizatori rapizi heterozigoi
(hetMR), iar restul combinaiilor *2/*2, *2/*3, *3/*3 sunt metabolizatori leni (ML). Varianta
CYP2C19*2 este relativ comun la populaia caucazian, de aproximativ 15% spre deosebire de
varianta CYP2C19*3 prezent n proporie de 0,04%. Aceasta din urm este mult mai frecvent
in populaiile asiatice si la afro-americani (0,4% i 5 %)
Fenitoina, un antiepileptic utilizat la scara larg, este un exemplu de medicament la care
apar diferene interindividuale de farmacocinetic, atribuite in mare msur unor factori genetici
cum ar fi polimorfismul CYP2C9. Fenitoina este metabolizat n mare msur de enzima
CYP2C9 i o parte i de enzima CYP2C19 rezultnd metabolitul 5-(parahidroxifenil)-
fenilhidantoin. Reducerea semnificativ a cleareance-ului fenitoinei (pn la 40% in cazul
mutaiilor genei CYP2C9 fa de varianta slbatic) a fost observat i in vivo la pacieni aflai
sub tratament antiepileptic , demonstrndu-se c mutaia CYP2C9 (n special alela *3) este
asociat cu risc crescut de apariie a toxicitii neurologice induse de fenitoin . n cazul
mefenitoinei, defectele de metabolizare pot fi atribuite la mai mult de 10 alele ale genei care
exprim CYP2C19.
n cazul diazepamului i a fenitoinei, primul metabolizat de CYP2C19, al doilea fiind
substrat al CYP2C9, s-a observat o diferen de maxim 2 ori a cleareance-ul hepatic sau a ariei
de sub curb (AUC) a concentraiei plasmatice n funcie de timp ntre metabolizatorii leni i
metabolizatorii rapizi. n cazuri rare, acidul valproic poate fi metabolizat ntr-un produs
hepatotoxic, acid 4-ene-valproic. Datele existente nu lmuresc deocamdat dac variantele
genetice ale enzimei implicate CYP2C9 sunt responsabile pentru aceast problem. n acelai
mod, metabolitul activ al carbamazepinei, carbamazepin-10,11-epoxide, este transformat de
epoxid hidroxilaza microzomial, o enzima care este de asemenea nalt polimorfic, dar
farmacocinetica i consecinele clinice vor trebui evaluate . Clobazamul este o benzodiazepin
utilizat n tratamentul epilepsiei refractare, la care principalele ci de metabolizare sunt
demetilarea de ctre CYP3A4 i CYP2C19, precum i hidroxilarea metabolitului activ N-
desmetilclobazam de ctre CZP2C19. CYP 2C19 este implicat n metabolismul hexobarbitalului,
mefobarbitalului i a fenobarbitalului n variate grade, ns aceast cale este responsabil doar de
o mic parte a biotransformrii i eliminrii fenobarbitalului. Mefobarbitalul urmeaz o cale de
metabolizare stericoselectiv. R-mefobarbitalul este metabolizat prin CYP2C19, iar un studiu
japonez a demonstrat o cretere de 92 de ori a ariei de sub curb a concentraiei n funcie de
timp la metabolizatorii leni fa de metabolizatorii rapizi, timpul de njumtire fiind de
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asemenea prelungit. Aceast diferen enorm poate constitui un motiv suficient care s justifice
genotiparea pe scar larg a pacienilor epileptici, mai ales a celor cu forme multirezistente.
Glicoproteina P (P-gp) acioneaz ca i o pomp membranar activ prin hidroliza ATP-
ului, dirijnd fluxul substanelor substrat specifice mpotriva unui gradient de concentraie.
Localizrile P-gp sugereaz rolul deinut de aceste proteine, acela de detoxifiere i protecie a
organismului fa de metaboliii i xenobioticele toxice, prin excreia acestora n bil, urin, n
lumenul intestinal i prin prevenirea acumulrii lor n creier, testicule i n organismul ftului. P-
gp este codificat de o mic familie de gene, ce reunete la om 2 gene denumite MDR1
(ABCB1) i MDR2 (ABCB4). Aceste gene sunt situate una lng cealalt pe cromozomul 7
(7q21) . Gena MDR1 codific transportorul specific pentru medicamente responsabil de
fenotipul polirezistenei medicamentoase i gena MDR2 codific un transportor pentru
fosfatidilcolin la nivelul canaliculilor biliari.
Epilepsia refractar la tratament se asociaz cu o supraexprimare a proteinelor
transportoare la nivel cerebral, fapt dovedit prin mai multe studii in vivo i in vitro. Patologia
lobului temporal a fost cel mai studiat focar epileptogen din punctul de vedere al reglrii
transportului medicamentelor la nivel cerebral. Tishel i colab. au evideniat o cretere de 10 ori
a ARNm MDR1 n esutul cerebral extirpat, la 11 din 19 pacieni la care s-a practicat lobectomie
temporal, comparativ cu esut cerebral normal. Dombrovski i colaboratorii au decelat o
supraexprimare a MDR1, MRP2 i MRP5 n celulele endoteliale izolate din vasele temporale ale
unor probe de esut prelevate de la 5 pacieni cu epilepsie refractar, tratat prin lobectomie
temporal.
O problem important este mecanismul prin care se ajunge la aceast supraexprimare a
transportorilor de eflux la nivel cerebral. Supraexprimarea transportorilor n creierul pacienilor
cu epilepsie poate fi constitutiv, dobndit sau mixt, prin asocierea acestora. Supraexprimarea
constitutiv poate aprea ca i rezultat al unei predispoziii genetice sau poate i intrinsec
dezvoltrii unei patologii specifice. Forma dobndit poate aprea n contextul unor crize
epileptice frecvente sau poate fi indus de antiepilepticele utilizate n scopul prevenirii crizelor.
Pn n prezent majoritatea studiilor au demonstrat implicarea ambelor mecanisme.
Testarea ipotezei genetice a rezistenei medicamentoase a fost realizat ntr-un studiu
amplu de ctre Siddiqui. S-a realizat genotiparea a 315 pacieni de ras caucazian diagnosticai
cu epilepsie cronic (200 de pacieni cu epilepsie rezistent la tratament i 115 responsivi) i 200
de pacieni fr epilepsie. Pacienii cu form refractar de boal au prezentat n procent mai mare
genotipul 3435 CC dect genotipul 3435TT, comparativ cu pacienii responsivi sau cu lotul
control . Asocierea rspunsului la terapia farmacologic i genotipul C3435T observat n aceste
studii nu a fost confirmat n altele care au inclus pacieni cu caracteristici etnice similare. O
meta-analiz a 11 studii (3371 de pacieni din care 1646 de pacieni cu epilepsie refractar i
1725 pacieni de control) care au urmrit influena mutaiei C3435T asupra epilepsiei refractare,
a demonstrat c nu exist diferene n privina frecventei mutaiilor la populaia refractar la
tratament i populaia care rspunde la tratament. Studiile privind disponibilitatea
medicamentelor asociate cu polimorfisme prezint n cele mai multe cazuri date discordante
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explicabile de heterogenitatea haplotipurilor n diferitele populaii i numrul relativ redus de
probe analizate, precum i datorit studierii unui numr mic de mutaii (n special a
polimorfismului C3435T). Studierea mai multor gene candidate i analiza multivariabil a
rezultatelor este direcia de urmat.

CONTRIBUIA PERSONAL

n aceast tez am evaluat influena polimorfismelor genelor care codific proteina de
eflux P-gp (MDR1 sau ABCB1) precum i a unor enzime de metabolizare (CYP2C9 i
CYP2C19) asupra frecvenei epilepsiei idiopatice sau secundare, asupra concentraiilor
plasmatice ale medicamentelor antiepileptice i asupra rspunsului la tratamentul antiepileptic
farmacologic.

Studiul 1 - Evaluarea frecvenei polimorfismelor genelor CYP2C9, CYP2C19 i MDR1 la
epileptici
Obiectivul principal al acestui studiu au fost evaluarea frecvenelor de apariie ale
polimorfismelor ABCB1 (MDR1) C3435T, G2677T/A i a T129C precum i a CYP2C9 i
CYP2C19 la pacieni cu epilepsie i compararea datelor obinute cu frecvenele acestor variante
la populaia de control. Secundar, am comparat frecvenelor de apariie ale genotipurilor MDR1,
CYP2C19 i CYP2C9 n populaia din Romnia cu frecvenele ntlnite n alte populaii
caucaziene i asiatice.
Studiul a evaluat 101 de pacieni diagnosticai cu epilepsie idiopatic sau secundar
conform criteriilor stabilite ILAE la acea dat. Am folosit ca i grup de control 126 de voluntari
sntoi pentru MDR1 G2677T/A i T-129C, petru restul date publicate anterior de la voluntari
sntoi din aceeai regiune geografic ca i lotul cu epilepsie.
S-au studiat patru polimorfisme ale genei MDR1 pentru proteina de eflux P-gp (C3435T,
T129C, G2677A i G2677T), 2 polimorfisme pentru CYP2C9 i respectiv 2 polimorfisme ale
CYP2C19.
Frecvenele de apariie ale mutaiilor CYP2C9 respectiv CYP2C9*2 (C416T) i
CYP2C9*3 (A1061C)9, nu au fost semnificativ diferite n lotul pacienilor epileptici fa de
populaia control. Frecvena de apariie a variantelor CYP2C19 studiate respectiv CYP2C19*1,
CYP2C19*2 i CYP2C19*3 nu au fost semnificativ diferite n lotul pacienilor epileptici fa de
populaia control.
Au fost determinate pentru prima oar pe o populaie din Romnia, frecvenele
polimorfismelor pentru mutaiile MDR1 G2677T/A i T-129C. Genotipurile MDR1 G2677T/A a
avut o frecven de apariie de 37% pentru varianta slbatic GG, 45% pentru heterozigoii GT,
15% pentru homozigoii mutani TT, 2% pentru heterozigoi GA i respectiv 1% pentru TA.
Genotipurile T-129C al genei MDR1 a avut o frecven de apariie de 95% pentru forma
homozigot slbatic TT i de 5% pentru heterozigot TC. Nu au fost depistate forme homozigote
mutante CC.
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Comparnd frecvenele MDR1 G2677T/A obinute n acest studiu pe lotul de subieci
sntoi cu alte rezultate obinute att pe populaie european ct i asiatic, nu au fost remarcate
diferene semnificative statistic ntre populaia din Romnia i celelalte populaii europene, dar
au fost observate diferene semnificative comparativ cu populaia asiatic. Aceste date corespund
observaiilor anterioare privind frecvena MDR1 G2677T/A. n privina studiului efectuat pe
mutaia T-129C , a fost observat o frecven mai mare a genotipurilor heterozigote n populaia
asiatic. Nu au fost remarcate diferene semnificativ statistic comparativ cu populaii europene
evaluate n alte studii. Comparnd frecvenele genotipurilor 2677 din studiul nostru s-a observat
o frecven mai sczut a variantei slbatice GG la pacienii epileptici fa de control, dar fr a
avea semnificaie statistic. Compararea frecvenelor alelelor MDR1 G2677T la pacienii cu
epilepsie idiopatic, respectiv epilepsie secundar cu lotul de control nu a demonstrat o diferen
semnificativ statistic pentru niciunul dintre grupuri, cu excepia alelei A, mai frecvent la lotul
cu epilepsie idiopatic.
Comparnd frecvena formei heterozigote TC a T-129C n funcie de tipul de epilepsie
remarcm o diferen ntre frecvena mai mare a genotipului la cei epilepsie secundar, fa de
cei cu epilepsie idiopatic. Avnd n vedere c aceast diferen nu este semnificativ, prin
compararea lotului cu epilepsie secundar cu lotul de control, acest rezultat poate fi datorat
probabil erorilor date de faptul c lotul studiat este mic i frecvena apariiei mutaiei este de
asemenea redus.

Studiul 2 - Corelarea concentraiilor de valproat cu polimorfismele CYP2C9, CYP2C19 i
MDR1
Acest studiu evalueaz variaiile concentraiilor plasmatice ale unui medicament
antiepileptic, acidul valproic, n funcie de polimorfismele genelor pentru enzimele microzomale
CYP2C9, CYP2C19 i pentru proteina transportoare P-gp (MDR1) pentru a putea face o
eventual corelaie ntre concentraiile plasmatice i anumite mutaii genetice. Secundar se
determin corelaia dintre rspunsul la tratament i concentraiile plasmatice ale acidului
valproic.
Studiul a evaluat 74 de pacieni epileptici aflai sub tratament cu acid valproic
(medicament antiepileptic unic sau asociat cu alte medicamente antiepileptice), pacieni aflai la
steady-state (doz constat de aprox o lun) Acestor pacieni li s-au determinat concentraiile
serice ale valproatului i polimorfismele genetice ale CYP2C9, CYP2C19 i MDR1.
Din cei 74 de pacieni evaluai n studiu, 43 (58,1%) au fost femei i 31 (41,9%) brbai.
mprirea n funcie de tipul de epilepsie a artat o frecven de 62,2% (46 de cazuri) de
epilepsie idiopatic i 37,8% (28 de cazuri) de epilepsie secundar. Tipul de epilepsie nu se
coreleaz cu vrsta, indicele de mas corporal, suprafaa corporal, sau cu sexul pacienilor.
S-au observat diferene semnificative statistic ale dozelor utilizate, ale dozei pe suprafaa
corporal (D/SC), precum i a raportului dintre dozele utilizate i concentraia plasmatica a
valproatului (D/C) ntre pacienii cu monoterapie i cei cu politerapie antiepileptic, n sensul
utilizrii unor doze mai mari la pacienii cu politerapie.
O cincime din pacienii studiai au avut concentraii plasmatice ale valproatului sub limita
inferioar terapeutic de 50 mg/l, dar un singur pacient non-responder (3,5% din totalul de non-
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responderi) a avut valori foarte reduse ale concentraiei plasmatice a valproatului (2,06 mg/l). La
ceilali 5 pacieni non-responderi cu concentraia plasmatic sub 50 mg/l, concentraiile
plasmatice s-au situat n jurul valorii de 40 mg/l. Cu toate acestea jumtate din pacieni au fost
ncadrai ca non-responder. Putem, deci, presupune c mecanismele care determin rezistena la
aceti pacieni sunt mult mai complexe dect simpla neatingere a concentraiilor considerate
terapeutice. De fapt doar un numr de 3 pacieni non-responder au concentraii supraterapeutice.
Dar avnd n vedere c, statistic, nu avem diferene semnificative ntre mediile concentraiilor
plasmatice la responder i non responder(p=0,36), putem presupune c, n cazul loturilor studiate
de noi, mecanismul de apariie a rezistenei nu depinde n mare msur de concentraiile
plasmatice ale valproatului. Dozele de valproat utilizate la non-responderi sunt semnificativ mai
mari fa de responder (p<0,001). Raportul doza utilizat pe concentraie este de asemenea
modificat semnificativ la pacienii non-responderi (p=0,001)
Concentraiile plasmatice ale valproatului nu difer semnificativ ntre loturile de pacienti
cu politerapie i cu monoterapie (p=0,939) precum i nici ntre cei cu epilepsie idiopatic i cei
cu epilepsie secundar (p=0,581)
Prezenta alelei C pentru 129 reduce semnificativ statistic concentraiile plasmatice ale
valproatului pn la valori subterapeutice, ns alte mutaii observate pentru MDR1 C3435T,
G2677T, CYP2C19 nu influeneaz semnificativ concentraiile plasmatice ale acidului valproic
i a raportului doz pe concentraie. n schimb, genotipul CYP2C9*2/*3 crete concentraia
valproatului pn la valori supraterapeutice (dar fr semnificaie statistic)

Studiul 3 - Studiul polimorfismelor genei MDR1 n epilepsia refractar

Ultimul studiu al tezei cerceteaz influena polimorfismelor MDR1 C3435T, G2677T/A i
T-129C ale genei transportorului membranar P-gp, asupra rspunsului la terapia antiepileptic.
Adiacent s-a efectuat analiza n lotul studiat a unor parametrii asociai , ca durata medie a bolii,
tipul de epilepsie.
n studiu au fost inclui 70 de pacieni diagnosticai cu epilepsie, aflai pe un regim stabil
de tratament antiepileptic de minim o lun. Pacienii au fost urmrii timp de un an, cu minim
patru vizite la medic. La sfritul acestei perioade s-a stabilit dac pacienii rspund sau nu la
tratament. Responderii sunt pacienii fr nici o criz epileptic timp de un an sub tratament
antiepileptic. Au fost exclui din studiu pacienii cu rspuns terapeutic nedefinit (potrivit
criteriilor ILAE), adic pacienii la care rspunsul la terapia adecvat nu poate fi definit nici ca
refractar (responder), nici cu rspuns pozitiv la tratament, non-responder (de exemplu pacienii
care post intervenie au ncetat s mai aib crize, dar nu este nc satisfcut criteriul de timp sau
pacienii care mai au crize , dar s-a ncercat numai o schem de tratament). Pentru comparare, s-a
utilizat un lot martor de voluntari sntoi.
n acest studiu s-au urmrit trei polimorfisme ale MDR1, respectiv C3435T, G2677T/A i
T-129C, dar nu s-a reuit demonstrarea unei asocieri ntre prezena anumitor polimorfisme i
epilepsia refractar la tratament n loturile studiate . Genotipurile C3435T al genei MDR1 au
avut o frecven de apariie la cazurile de pacieni epileptici de 20% pentru homozigot slbatic
CC, de 54,3% pentru hetrozigoii CT i 25,7% pentru homozigoii mutanii CC. Genotipurile
MDR1 G2677T/A a avut o frecven de apariie de 32,9% pentru varianta slbatic GG, 47,1%
pentru heterozigot GT, 14,3% pentru homozigot mutant TT, 1,4% pentru heterozigot GA i
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respectiv 4,3% pentru TA . Genotipurile T-129C al genei MDR1 a avut o frecven de apariie de
92,9% pentru forma homozigot slbatic TT i de 7,1% pentru heterozigot TC. Nu au fost
depistate forme homozigote mutante CC. Prezena polimorfismelor C3435T, G2677T/A i T-
129C ale genotipului MDR1 (ABCB1) la pacienii epileptici din loturile studiate nu a fost
asociat cu rspunsul la tratamentul cu medicamente antiepileptice, avnd frecvene de apariie
care nu difer semnificativ la pacienii responderi i la cei nonresponderi.
52,86% din de pacienii epileptici inclui n studiu nu au avut un rspuns adecvat la
tratamentul antiepileptic. Frecvena pacienilor non-responderi este mai crescut dect n studiile
care arat o frecven de aproximativ 30-35% a epilepticilor care nu rspund la tratament.
Aceasta se datoreaz probabil unei adresabilitii mai mari a acestor pacieni la un centru
regional cum este Clinica de Neurologie a Spitalului Judeean Cluj.
A fost observat i o medie a duratei bolii semnificativ crescut pentru pacienii non-
responderi (p=0,0032) care poate fi explicat de asemenea printr-o adresabilitate mai mare ctre
Clinica de Neurologie a pacienilor care au probleme n a controla prin medicaie crizele
epileptice. Exis o diferen semnificativ statistic i ntre durata boli n cazul epilepsiei
idiopatice fa de epilepsia secundar, n sensul creterii duratei bolii pentru epilepsia idiopatic
(p=0,042). Avnd n vedere c nu exist diferene semnificative ntre mediile vrstei celor dou
categorii de pacieni, probabil aceast diferen este dat de debutul epilepsiei idiopatice la o
vrst mai mic n raport cu epilepsia secundar.

Concluzii generale

1. Mutaiile MDR1 G2677T/A i T-129C, au fost determinate pentru prima oar pe o
populaie din Romnia,
2. Frecvenele polimorfismelor CYP2C9, CYP2C19 i MDR1 detectate nu sunt diferite fa
de ale studii efectuate pe alte populaii caucaziene europene, dar semnificativ diferite fa
de frecvenele din populaii asiatice.
3. Alela A pentru MDR1 G2677T/A, mai frecvent n populaia cu epilepsie idiopatic, n
rest nu sunt diferene ale frecvenelor mutaiilor CYP 2C9, CYP2C19, MDR1 studiate n
populaia epileptic general fa de populaia control.
4. Pentru genotipul MDR1 -129 TC s-au observat frecvene mai crescute la pacienii cu
epilepsie secundar comparativ cu cei cu epilepsie idiopatic.
5. O cincime din populaia studiat a avut valori ale concentraiilor valproatului sub valorile
minime terapeutice (<50 mg/l)
6. Prezenta alelei C pentru 129 reduce semnificativ statistic concentraiile plasmatice ale
valproatului pn la valori subterapeutice.
7. Genotipul CYP2C9*2/*3 crete concentraia valproatului pn la valori supraterapeutice
(dar fr semnificaie statistic)
8. Nu au fost evideniate asocieri ntre polimorfismele C3435T, G2677G/A i T-129C ale
MDR1 (ABCB1) i rspunsul la tratamentul cu medicamente antiepileptice
9

Summary of the PhD thesis

IMPLICATIONS OF MDR1, CYP 2C9 AND CYP 2C19 GENE POLYMORPHISMS IN
REFRACTORY EPILEPSY

PhD candidate: Octavia Sabin
Scientific Coordinator: Prof. Dr. Anca-Dana Buzoianu


Keywords: refractory epilepsy, efflux protein, drug-metabolizing enzymes, genetic
polymorphism, MDR1, CYP2C9, CYP2C19

TABLE OF CONTENTS

INTRODUCTION 13
CURRENT KNOWLEDGE
1. Epilepsy definition, classification 17
2. Epilepsy pharmacogenetics 19
2.1. Hypotheses on the causes of refractory epilepsy 20
2.2. Variability of CYP 450 system 21
2.3. P Glycoprotein
28
PERSONAL CONTRIBUTION
1. Research hypothesis/general objectives 39
2. General methodology 39
3. Study 1 Evaluation of CYP2C9, CYP2C19 and MDR1
polymorphism frequency in epilepsy populations
47
4. Study 2 Correlation between valproic acid concentrations
and CYP2C9, CYP2C19 and MDR1 polymorphisms
69
5. Study 3 Study of MDR1 polymorphism in refractory epilepsy 101
6. General conclusions (summary) 119
7. Original and innovative contributions of the thesis 121
REFERENCES 123
10
CURRENT KNOWLEDGE

Pharmacogenomics analyses how human genome variations influence an individuals
response to drugs. It allows adaptation of medication to the individuals specific genetic markup
by means of individualized treatment options, optimized for each patient, highly effective and
secure. The first steps were taken with the analysis of specific mutations in certain types of
cancer, which made possible the use of specific cancer drugs. In particular cases, genotype
analysis is an absolutely necessary step before prescribing drug therapy. However, it seems to be
a promising clinical approach in the treatment of other diseases as well, to minimize drug side
effects and increase drug efficiency.
Epilepsy is a group of long-term neurological disorders characterized by repeated
epileptic seizures. More than 50 million people in the world have epilepsy (incidence of 0.5-1%),
characterized by recurrent episodes of critical or non-critical seizures, determined by focal or
generalized nerve cell activity in the brain. Seizures are controllable with medication in over
two-thirds of the patients, but there is still a large number of people in which epileptic crises are
difficult to control, in spite of well-managed and adequate antiepileptic therapy. Refractory
forms of epilepsy are associated with high mortality and debilitating psycho-social
consequences.
One of the most common hypotheses is the modification of antiepileptic drug
pharmacokinetics, for instance the variability of drug-metabolizing enzyme affinity. Mutations in
CYP genes can eliminate, reduce, alter or increase enzyme activity, respectively more individual
phenotype expressions: slow, intermediate, rapid and ultra-rapid metabolizers. By obtaining
lower than therapy-level plasma concentrations, the therapeutic response can be unsatisfactory.
In patients with adequate plasma concentrations, several hypotheses have been issued
concerning the etiology of multidrug resistance to antiepileptic therapy. The main two concepts
attempting to explain this multidrug resistance are 1) modification of action target and 2)
modification of efflux protein function. The first hypothesis is based on studies showing that
certain structural modifications or anomalies in the neuronal network and in the activity of
certain neurotransmitters occurring in epileptic patients can be caused by resistance to
antiepileptic drug therapy, i.e. the disorder is characterized by the modification of the action
target. The second hypothesis resulted from the observation that one of the main obstacles in
treating neurological disorders, including epilepsy, is the weak penetration through the
physiological barrier separating the brain tissue from the blood, i.e. the blood-brain barrier
(BBB). In addition to the very tight intercellular junctions, there is a high density of ABC
transporters at BBB, which determines multiple drug resistance. Starting from this idea and from
the observation that refractory epilepsy patients are non-responsive to several antiepileptic drugs
with different clinical mechanisms of action, a non-specific mechanism was suggested, which
limits antiepileptic drug efficiency by over-expressing efflux transporters at BBB. The prototype
of these transporters is the P glycoprotein (P-gp).
The cytochrome P450 enzymes in the liver and gastrointestinal tract have a long history of
pharmacogenetic studies, which demonstrated a clinical relevance of polymorphisms, and
especially CYP2C9, CYP2C19, and CYP2D6 polymorphisms. CYP2C9 is involved in the
metabolism of more than 100 drugs, including coumarin oral anticoagulants, sulphonylureas,
11
angiotensin inhibitors and some nonsteroidal anti-inflammatory drugs. In-vitro studies have
shown that CYP2C9*3 is associated with higher decrease in intrinsic clearance as compared to
CYP2C9*2. At least one of the CYP2C9*2 or *3 variants is present in approximately 20%,
respectively 12% of the Caucasian population, approximately 2.5 % are homozygous for these
alleles or heterozygous type *2/*3. The remaining two-thirds of the Caucasian population show
normal enzyme activity. For CYP2C19, homozygous for the wild type alleles in exons 5 and 4
(1*/1*) were classified as homozygous rapid metabolizers (homRM), mutations of type *1/*2,
*1/*3 as heterozygous rapid metabolizers (hetRM), and the remaining combinations *2/*2,
*2/*3, *3/*3 are slow metabolizers (SM). The CYP2C19*2 variant is relatively common in the
Caucasian population, with approximately 15% versus the CYP2C19*3 variant, with 0.04%. The
latter is significantly more frequent in Asian and Afro-American populations (0.4% and 5%)
Phenytoin, a widely-used antiepileptic drug, is an example of inter-individual
pharmacokinetic variation, largely due to genetic factors, such as CYP2C9 polymorphism.
Phenytoin is primarily metabolized by the CYP2C9 enzyme and partially by the CYP2C19
enzyme, to 5-(parahydroxyphenyl)-phenylhydantoin. Significant reduction in phenytoin
clearance (up to 40% for the CYP2C9 mutations as opposed to the wild variant) was also
observed in vivo, in patients receiving antiepileptic therapy, which proves that the CYP2C9
mutation (especially allele *3) is associated with an increased risk of phenytoin toxicity. In
mephenytoin, the metabolism defects can be attributed to more than 10 alleles of the CYPC19
gene.
In diazepam and phenytoin, the first metabolized by CYP2C19 and the second being a
CYP2C9 substrate, a maximum two-fold difference was observed in liver clearance or area under
the curve (AUC) of plasmatic concentration, depending on the time between slow and rapid
metabolizers. In rare instances, valproic acid can be metabolized in a hepatotoxic product, 4-ene-
valproic acid. The existing data is currently inconclusive if this issue is caused by genetic
variants of the CYP2C9 enzyme. Similarly, the active metabolite of carbamazepine,
carbamazepin-10,11-epoxyde, is transformed by microsomal epoxide hydroxylase, an enzyme
which is highly polymorphic, but the pharmacokinetics and the clinical consequences are still to
be evaluated. Clobazam is a benzodiazepine used in refractory epilepsy therapy. Its major
metabolizing pathway involves CYP3A4 and CYP2C19 demethylation, as well as hydroxilation
of N-desmetylclobazam active metabolite by CYP2C19. CYP2C19 is involved in the metabolism
of hexobarbital, mefobarbital and phenobarbital to different degrees, but this pathway is only
partially responsible for phenobarbital biotransformation and elimination. Mefobarbital follows a
stereoselective metabolizing pathway. R-mefobarbital is metabolized by CYP2C19. A Japanese
study demonstrated a 92-fold increase in the time-dependent area under the curve of the
concentration at slow metabolizers versus rapid metabolizers, and half time also extended. This
enormous difference can constitute in itself a justification for large-scale genotyping of epileptic
patients, especially those with multi-resistant forms of epilepsy.
P-glycoprotein (P-gp) acts as an active membrane pump by ATP hydrolysis, by guiding the
flow of specific substrate substances against a concentration gradient. P-gp locations indicate the
12
role of these proteins in cleaning and protecting the body from toxic metabolites and xenobiotics,
by eliminating these substances in the bile, urine, intestinal lumen and preventing their
accumulation in the brain, testicles and fetus. P-gp is coded by a small family of genes, which in
humans comprise 2 genes called MDR1 (ABCB1) and MDR2 (ABCB4). These genes are located
next to each other on chromosome 7 (7q21). MDR1 gene mediates the specific drug transfer
involved in the phenotype of multidrug resistance and MDR2 gene mediates a
phosphatidylcholine transfer in biliary vesicles.
Refractory epilepsy is associated with an overexpression of drug transport proteins in the
brain, which has been demonstrated by several in vivo and in vitro studies. The pathology of the
temporal lobe was the most frequently studied cause of epilepsy involved in the regulation of
drug transport in the brain. Thisel et al. indicated a 10-fold ARNm MDR1 increase in extirpated
brain tissue in 11 out of 19 patients subject to temporal lobectomy as compared to normal brain
tissue. Dombrovski et al. determined an overexpression of MDR1, MRP2 and MRP5 in
endothelial cells isolated from temporal lobe blood vessels of 5 patients with refractory epilepsy,
treated by temporal lobectomy.
An important issue is the mechanism of this overexpression of efflux transporters in the
brain. Overexpression of transporters in the brain of epilepsy patients can be genetic, acquired or
a combination of the two. Genetic overexpression can occur as a result of genetic predisposition
or may be caused by specific pathology. The acquired form can manifest in the context of
frequent epileptic seizures or can be triggered by antiepileptic drugs taken as a seizure prevention
measure. Most of the studies performed until now have demonstrated that both mechanisms are
involved.
The genetic hypothesis of drug resistance was tested in a large study performed by
Siddiqui. The study involved genotyping of 315 Caucasian patients diagnosed with chronic
epilepsy (200 patients with drug resistant epilepsy and 115 drug-responsive patients) and 200
control subjects without epilepsy. As opposed to the patients with drug-responsive epilepsy or
with the control group, patients with drug-resistant epilepsy were more likely to have the CC
genotype at 3435 than the TT genotype. The association between drug therapy response and
C3435T genotype observed in these studies was not confirmed by other studies including
patients with similar ethnic characteristics. A meta-analysis of 11 studies (3371 patients of which
1646 patients with refractory epilepsy and 1725 control subjects) examining the influence of
C3435T mutation on refractory epilepsy demonstrated that there are no differences in mutation
frequencies between the drug-refractory population and the drug-responsive population. Most of
the studies concerning drug availability associated with polymorphisms contain discordant data
explained by the heterogeneity of haplotypes in different populations and a relatively low
number of analyzed samples, but also the small number of studied mutations (especially C3435T
polymorphism). The direction to follow in the future is to study several candidate genes and
perform a multivariable analysis of the results.

PERSONAL CONTRIBUTION
13

In this thesis we assessed the influence of gene polymorphisms mediating the efflux
protein P-gp (MDR1 or ABCB1), and of metabolizing enzymes (CYP2C9 and CYP2C19) on the
frequency of idiopathic or secondary epilepsy, on plasma concentrations of antiepileptic drugs
and on the antiepileptic drug response.

Study 1 - Evaluation of CYP2C9, CYP2C19 and MDR1 polymorphism frequency in
epilepsy populations
The main objective of this study was to evaluate the occurrence frequency of
polymorphisms ABCB1 (MDR1) C3435T, G2677T/A and of T129C, as well as CYP2C9 and
CYP2C19 in epileptic patients and to compare the resulting data with the frequency of these
variants in the control group. Secondarily, we compared the occurrence frequency of MDR1,
CYP2C19 and CYP2C9 genotypes in Romanian population with the frequencies in Caucasian
and Asian populations.
The study evaluated 101 patients diagnosed with idiopathic or secondary epilepsy
according to the established ILAE criteria to that date. We used a control group of 126 healthy
subjects for MDR1 G2677T/A and T-129C, for the remaining data previously published from
healthy subjects in the same geographic area as the epilepsy group.
Four polymorphisms of MDR1 gene were studied for the efflux protein P-gp (C3435T,
T129C, G2677A and G2677T), 2 polymorphisms for CYP2C9 and 2 polymorphisms for
CYP2C19.
The frequency of CYP2C9, respectively CYP2C9*2 (C416T) and CYP2C9*3 (A1061C)9
mutation were not significantly different in the epilepsy group versus the control group. The
frequency of CYP2C19 variants, respectively CYP2C19*1, CYP2C19*2 and CYP2C19*3 were
not significantly different in the epilepsy group versus the control group.
The frequencies of polymorphism frequencies for MDR1 G2677T/A and T-129C
mutations were determined for the first time in a Romanian population. The frequency of MDR1
G2677T/A genotypes was 37% for the wild type GG, 45% for heterozygous GT, 15% for the
mutant homozygous TT, 2% for heterozygous GA, and 1% for TA. The frequency of MDR1 T-
129C genotypes was of 95% for the homozygous wild type TT and 5% for the heterozygous TC.
No homozygous mutant CC variants were detected.
A comparison between the MDR1 G2677T/A frequencies obtained in this study in the
control group and other results obtained both in European and Asian populations revealed no
statistically significant differences between the Romanian population and other European
populations. However, significant differences were observed in comparison with the Asian
population. These conclusions are similar to previous observations concerning MDR1 G2677T/A
frequency. As concerns the study on T-129C mutation, a higher frequency of heterozygous
genotypes was observed in the Asian population. No statistically significant differences were
determined as compared to European populations evaluated in other studies. A comparison
between the 2677 genotypes in our study revealed a lower frequency of wild-type GG in
epileptic patients versus the control group, but with no statistical significance. A comparison
14
between MDR1 G2677T allele frequencies in patients with idiopathic respectively secondary
epilepsy and the control group revealed no statistically significant difference for none of the
groups, except for allele A, which is more frequent in the idiopathic epilepsy group.
A comparison between the frequency of the heterozygous variant TC a T-129C in various
epilepsy types reveals a higher frequency of the genotype in patients with secondary epilepsy
versus idiopathic epilepsy. Given that this difference is not significant when comparing the
secondary epilepsy group with the control group, the result is probably erroneous due to the
small size of the group and the low frequency of the mutation.

Study 2 - Correlation between valproic acid concentrations and CYP2C9, CYP2C19 and
MDR1 polymorphisms
This study examines the variations between plasma concentrations of valproic acid, an
antiepileptic drug, based on gene polymorphisms in microsomal enzymes CYP2C9, CYP2C19
and the transporter protein P-gp (MDR1) to make a correlation between plasma concentrations
and certain genetic mutations. Secondarily, the correlation between the treatment response and
the plasma concentrations of valproic acid is determined.
The study evaluated 74 epileptic patients receiving therapy with valproic acid (an unique
antiepileptic drug associated with other antiepileptic drugs), patients in steady state (constant
dose for approximately one month). These patients were tested for serum concentrations of
valproic acid and genetic polymorphisms of CYP2C9, CYP2C19 and MDR1.
The study comprised 74 patients, of which 43 (58.1%) women and 31 (41.9%) men. The
classification based on epilepsy type indicated a 62.2% frequency (46 cases) of idiopathic
epilepsy and 37.8% (28 cases) of secondary epilepsy. The type of epilepsy is not correlated with
age, body mass index, body surface area or gender.
Statistically significant differences in the doses, dose per body surface area (D/BSA) and
ratio of dose to valproic acid plasma concentration (D/C) were observed in the patients receiving
single-drug versus multidrug antiepileptic therapy, i.e. higher dosing was used in patients
receiving multidrug therapy.
A fifth of the patients in the study had valproic acid plasma concentrations lower than the
minimum therapy values of 50 mg/l, but only one non-responder (3.5% of the total number of
non-responders) had very low valproic acid plasma concentrations (2.06 mg/l). In the other 5
non-responding patients with plasma concentrations under 50 mg/l, plasma concentration values
were at approximately 40 mg/l. However, half of the patients were considered non-responders.
Therefore, we can assume that the mechanisms determining resistance in these patients are much
complicated than the failure to reach the concentrations considered to be at a therapeutic level.
Actually only a number of 3 non-responders have concentrations above the therapy level.
Considering that there are no statistically significant differences between the average plasma
concentrations in responders and non-responders (p=0.36), we can assume that in the groups in
our study the resistance mechanism is not significantly dependent on valproic acid plasma
concentrations. Valproic acid doses given to non-respondents are significantly higher than the
doses given to responders (p<0.001). The dose per concentration ratio is also significantly altered
in non-responders (p=0.001)
15
Valproic acid plasma concentrations are not significantly different in patient groups
receiving single-drug versus multidrug therapy (p=0.939), also between patients with idiopathic
versus secondary epilepsy (p=0.581)
The presence of allele C for 129 determines a statistically significant decrease in valproic
acid plasma concentrations to lower than therapy values, but other observed mutations for MDR1
C3435T, G2677T, CYP2C19 do not significantly influence the valproic acid plasma
concentrations or the dose per concentration ratio. However, the CYP2C9*2/*3 genotype
increases valproic acid concentrations to higher than therapy values (but not statistically
significant).

Study 3 - Study of MDR1 polymorphism in refractory epilepsy

The last study of the thesis examines the influence of MDR1 C3435T, G2677T/A and T-
129C polymorphisms, the gene of the P-gp membrane transporter, on the antiepileptic drug
response. Additionally, an analysis of associated parameters, such as average duration of the
disorder or the type of epilepsy was performed.
The study included 70 patients diagnosed with epilepsy on a stable drug regimen of at
least one month. The patients were monitored for a year, with minimum of four follow-up up
visits. At the end of this period, the patients were classified as responsive or non-responsive to
treatment. Responders are patients having no epilepsy seizures for a year of antiepileptic therapy.
The study did not include patients with undefined therapy response (according to ILAE criteria),
i.e. patients in which response to adequate therapy cannot be defined as refractory (responder),
with a positive response to therapy, non-responders (for instance patients who ceased to have
seizures after the intervention, but the time criterion is not fulfilled yet or patients who still have
seizures, but only one therapy plan was attempted). A control group of healthy subjects was used
for comparison.
The study monitored three MDR1 polymorphisms, C3435T, G2677T/A and T-129C, but
could not demonstrate an association between the presence of certain polymorphisms and drug
refractory epilepsy in the studied groups. The MDR1 C3435T genotype frequencies in epileptic
patients were of 20% for homozygous wild-type CC, 54.3% for heterozygous CT and 25.7% for
homozygous mutant CC. The frequency of MDR1 G2677T/A genotypes was of 32.9% for the
wild-type GG, 47.1% for heterozygous GT, 14.3% for the mutant homozygous TT, 1.4% for
heterozygous GA, and 4.3% for TA. The frequency of MDR1 T-129C genotypes was of 92.9%
for the homozygous wild-type TT and 7.1% for the heterozygous TC. No homozygous mutant
CC variants were detected. The presence of MDR1 (ABCB1) genotype C3435T, G2677T/A and
T-129C polymorphisms in epilepsy patients in the studied groups was not associated with the
antiepileptic drug therapy response, as the occurrence frequencies were not significantly different
in responder versus non-responder patients.
Of the patients included in the study, 52.86% did not have an adequate response to
antiepileptic drug therapy. The frequency of non-responder patients is higher than in studies
indicating an approximate 30-35% frequency of non-responsive epileptic patients. This is
probably due to easier access of these patients to a regional clinical center, the Neurology Clinic
of the Cluj County Hospital.
16
The average duration of the disease was significantly longer in non-responsive patients
(p=0.0032), a fact that can also be attributed to easier access to the Neurology Clinic of the
patients with difficulties controlling epileptic seizures with drug therapy. There is also a
statistically significant difference between the duration of the idiopathic versus secondary
epilepsy, with an increased duration in the case of idiopathic epilepsy (p=0.042). Given that there
are no significant differences between the average ages of the two patient categories, this
difference is probably determined by the earlier onset of idiopathic epilepsy versus secondary
epilepsy.

General conclusions

9. MDR1 G2677T/A and T-129C mutations were determined for the first time in a
Romanian population.
10. The frequencies of CYP2C9, CYP2C19 and MDR1 polymorphisms do not differ from
other studies on other European Caucasian populations, but differ significantly from
frequencies in Asian populations.
11. Allele A for MDR1 G2677T/A is more frequent in idiopathic epilepsy population, but this
is the only difference in frequencies of CYP 2C9, CYP2C19, MDR1 mutations studied in
the general epileptic population versus the control group.
12. For the MDR1 -129 TC genotype higher frequencies were determined in patients with
secondary epilepsy versus patients with idiopathic epilepsy.
13. A fifth of the population in the study had valproic acid concentrations below minimum
therapy values (<50 mg/l).
14. The presence of allele C for 129 determines a statistically significant reduction in plasma
concentrations of valproic acid to lower than normal therapy values.
15. The CYP2C9*2/*3 genotype increases valproic acid concentrations to higher than normal
therapy values (but not statistically significant).
16. No associations were determined between C3435T, G2677G/A and T-129C of MDR1
(ABCB1) polymorphisms and the response to antiepileptic drug therapy.