UNIVERSITATEA DE MEDICIN I FARMACIE, TRGU MURE

Facultatea de Farmacie

Chimie General i Anorganic!

DEZVOLTAREA I OPTIMIZAREA UNEI METODE

MICROANALITICE MEDIATE ELECTROFORETIC PENTRU STUDII
DE METABOLISM: MONITORIZAREA ACTIVITII CYP1A1
!

Coordonator,

Conf. Dr. Augustin Curticpean

Absolvent,

Mnica Laura-Georgiana

Trgu Mure

2016
 UNIVERSITY OF MEDICINE AND PHARMACY, TRGU MURE

Faculty of Pharmacy

General and Inorganic Chemistry Department!

DEVELOPMENT AND OPTIMIZATION OF AN

ELECTROPHORETICALLY MEDIATED MICROANALYSIS SYSTEM
FOR METABOLISM STUDIES: APPLICATION TO CYP1A1
ACTIVITY MONITORING
!

Scientific Coordinator,

Assoc. Prof. Dr. Augustin Curticpean

Graduate student,

Mnica Laura-Georgiana

Trgu Mure

2016
 ACKNOWLEDGEMENTS

I would like to express my gratitude to my coordinator, Assoc. Prof. Dr.

Augustin Curticpean, for his full support, patience, encouragement, and understanding
throughout my study and research. His extensive knowledge and vision have been a
great source of inspiration for me.

I would also like to express my deepest appreciation to Prof. Dr. Marianne Fillet
and Prof. Dr. Anne-Catherine Servais, for giving me the chance to work in a modern
research environment, during my Erasmus + Scholarship. Without their expert guidance
and professionalism, this paper would not have existed.

I am especially indebted to my supervisor, Elena Farca, for her invaluable

advice and kindness. I could not have been more lucky to discover a true friend, in the
person of a great mentor.

Finally, I would like to thank my family and friends, for being so supportive
during this period of time.
 CONTENTS

ABBREVIATIONS __________________________________________________ i
REZUMAT _______________________________________________________ I-XII
I.GENERAL PART __________________________________________________ 1
I.1. INTRODUCTION ________________________________________________ 1
I.2. METABOLISATION STUDIES ____________________________________ 2
I.2.1. Drug metabolism ____________________________________________ 2
I.2.2. CYP 1 family _______________________________________________ 3
I.2.3. In vitro metabolism studies ____________________________________ 4
I.3. CAPILLARY ELECTROPHORESIS ________________________________ 6
I.3.1. Introduction _________________________________________________ 6
I.3.2. In-capillary assays ____________________________________________ 6
I.3.3. Electrophoretically mediated microanalysis ________________________ 7
I.3.3.1. Theoretical backround _____________________________________ 7
I.3.3.2. EMMA modes ___________________________________________ 7
I.3.3.3. Development and optimization ______________________________ 9
I.3.3.3.1. Key factors ______________________________________ 9
I.3.3.3.2. Injection, separation, and detection ___________________ 10
II. EXPERIMENTAL PART ________________________________________ 13
II.1. INTRODUCTION ______________________________________________ 13
II.2. MATERIALS AND METHODS __________________________________ 14
II.2.1. Instrumentation and capillaries ________________________________ 14
II.2.2. Reagents and chemicals ______________________________________ 14
II.2.3. Buffer and sample preparation_________________________________ 15
II.2.4. EMMA procedure __________________________________________ 16
II.2.5. Offline incubation procedure _________________________________ 17
II.2.6. Inhibition assay procedure ___________________________________ 17
II.3. RESULTS AND DISCUSSION ___________________________________ 18
II.3.1. Preliminary studies inlet injection optimization _________________ 18
II.3.1.1. Development of a CE method for EC and HC separation ____ 18
II.3.1.2. Offline metabolization _______________________________ 19
II.3.1.3. Online metabolization ________________________________ 20
 II.3.1.4. Use of internal standards ______________________________ 21
II.3.1.5. Injection order _____________________________________ 24
II.3.1.6. Microsomes concentration ____________________________ 25
II.3.2. Preliminary studies outlet injection optimization ________________ 26
II.3.2.1. Injection order and voltage switch ______________________ 27
II.3.2.2. Incubation time _____________________________________ 28
II.3.2.3. Microsomes concentration ____________________________ 28
II.3.2.4. Voltage switch and switch time influence ________________ 29
II.3.3. Optimization final steps ___________________________________ 30
II.3.3.1. Optimization of incapillary metabolization procedure ______ 31
II.3.3.2. Study of the enzymatic parameters _____________________ 33
II.3.3.3. Design of the experiment ____________________________ 34
II.3.4. Applicability _____________________________________________ 36
II.3.4.1. Applicability to microsomes in vitro model ______________ 36
II.3.4.2. Applicability to CYP1A1 inhibitors screening ____________ 36
CONCLUSIONS ____________________________________________________ 38
REFERENCES ______________________________________________________ 40

!
 ABBREVIATIONS

NCE new chemical entity

CYPs cytochromes P450
NADPH nicotinamide adenine dinucleotide phosphate reduced
LC liquid chromatography
MS mass spectrometry
CE capillary electrophoresis
EMMA electrophoretically mediated microanalysis
BGE background electrolyte
TDLFP transverse diffusion of laminar flow profiles
RPS rapid polarity switch
MWNT multi-walled carbon nanotube
DME drug metabolizing enzyme
CZE capillary zone electrophoresis
MECK micellar electrokinetic chromatography
EOF electroosmotic flow
CMC critical micellar concentration
SDS sodium dodecyl sulfate
LIF laser-induced fluorescence
7-EC 7-ethoxycoumarin
7-HC 7-hydroxycoumarin
MO mesityl oxide
DoE design of experiment
IB incubation buffer
DMSO dimethyl sulfoxide

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 REZUMAT

Att eficacitatea ct i toxicitatea substanelor medicamentoase depind n mare

msur de proprietile farmacocinetice ale acestora. n acest context, o bun nelegere
a metabolismului entitilor chimice devine esenial n momentul dezvoltrii unui
medicament [2]. Studiile in vitro asupra metabolizrii noilor substane chimice au astzi
o importan crescnd, fiind posibil utilizarea lor nca din stadiul preclinic.
Metabolizarea unei substane poate fi definit ca un proces de conversie, de la o
molecul lipofil spre una hidrofil, ce poate fi eliminat cu mai mult uurin de ctre
organismul uman sau animal [3]. Principala clas de enzime cu rol n metabolizare este
cea a Citocromului P450 (CYP450), cu o pondere de 75% asupra biotransformrii
produselor medicamentoase aflate pe piaa farmaceutic [5].
n ultimii ani, a prezentat interes pentru lumea tiinific, o nou familie CYP i
anume CYP1, datorit polimorfismului i activitii reglatoare de care dau dovad
membrii acesteia. Este alctuit din izoenzimele CYP1A1, CYP1A2 i CYP1B1,
exprimate preponderent n esutul extrahepatic. Se regsesc cu precdere la nivelul
pancreasului, timusului, uterului sau al intestinului subire i joac un rol primordial n
metabolismul xenobioticelor [6]. CYP1A1 este de asemenea cunoscut ca fiind una
dintre cele mai importante enzime n bioactivarea unor procarcinogene [7]. Ca i
consecin, aceasta a devenit i o posibil int n tratamentul cancerului.
ns, pentru a putea detecta cu exactitate tipul enzimei implicate n procesul
biochimic, este necesar folosirea studiilor in vitro. Acestea pot fi realizate cu:
hepatocite proaspete sau criogenate, felii de esut hepatic, microzomi hepatici [3] sau
enzime recombinate (superzomi) [11]. Cele din urm, permit studiul unei singure
enzime de interes, specificitatea analizei crescnd considerabil. Dac la nceput, erau
obinute prin recombinarea proteinelor ce aparineau unor organisme artropode, astzi
biologia molecular permite clonarea i exprimarea unui numr vast de izoenzime
CYP450 umane, disponibile comercial [3].
O caracteristic important a microzomilor i superzomilor este compatibilitatea
acestora cu tehnicile moderne de analiz. Momentan, se ntlnesc trei tipuri de abordri:
sisteme biocatalitice offline, bazate pe marcarea fluorescent sau radioactiv a
metaboliilor, tehnici imunoenzimatice i sisteme biocatalitice online. Cele din urm au
avantajul cuplrii directe cu separarea i detectarea reactanilor, prin intermediul
tehnicilor precum cromatografia lichid sau electroforeza capilar [4].

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 n mod tradiional, electroforeza capilar era utilizat doar ca un instrument de
separare n cadrul analizelor metabolice, reacia avnd loc n afara tubului capilar [28].
Totui, acest abordare aduce cu sine o serie de limitri, cum ar fi consumul ridicat de
reactivi chimici sau un timp prelungit de analiz.
EMMA (Electrophoretically mediated microanalysis/ analiz mediat
electroforetic) s-a dovedit a fi o tehnic mult mai avantajoas pentru acest tip de
investigaii, lund n considerare eficiena sa nalt i timpul sczut de analiz ce poate
fi atins. Capilara devine astfel un microreactor, unde analiii interacioneaz, fiind
totodat separai i detectai, consumul de substane fiind redus la minim.
Utiliznd aceast abordare, Curcio et al. [5] au descris cu uurin activitatea
CYP450 folosind dextrometorfanul pe post de substrat enzimatic. Zhang et al. [39] , de
asemenea, au caracterizat activitatea CYP3A4, folosind testosteronul i nifedipina ca i
substrat. Mai mult, metabolizarea enantioselectiv a fluoxetinei pe calea CYP2D6 [40]
sau N-demetilarea stereoselectiv a verapamilului [41] i a ketaminei [42] au fost
analizate, confirmnd versatilitatea metodei.
Atenia cercettorilor s-a ndreptat i ctre investigarea factorilor ce influeneaz
procesul electroforetic, fiecare dintre acetia fiind studiat ns n mod individual. Dac
un grup de cercetare [43] a descris influena compoziiei tamponului de separare asupra
procesului metabolic, un altul [28] a supus examinrii importana realizrii unui schimb
rapid de polaritate n cadrul mixing-ului electroforetic. Un al treilea grup a reuit s
prezic overlap-ul plug-urilor injectate, printr-o simulare computerizat.
n acest lucrare, a fost dezvoltat o metod complet automatizat pentru
studierea activitii CYP1A1 [2]. 7-etoxicumarina a fost aleas ca i substrat, posednd
cea mai mic valoare Km pentru enzima utilizat (10 0.1M) [44]. n prezena
nicotinamid adenin dinucleotid fosfat redus ca i co-factor, 7-etoxicumarina (7-EC)
sufer o reacie de O-deetilare, dnd natere produsului de reacie, 7-hidroxicumarina
(7-HC), molecul ce va fi monitorizat n studiul de fa (Figura II.2.1.). Optimizarea
prin studierea simultan a multiplilor factori implicai n acest proces s-a fcut prin
aplicarea unui design experimental. Este pentru prima dat cnd un astfel de instrument
analitic este utilizat.

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 7- Etoxicumarina (EC)

H3C O O O

CYP1A1 + NADPH, 37C

7- Hidroxicumarina (HC)

HO O O

Figura II.2.1. Reacia enzimatic a transformrii EC n HC

Cele mai promitoare rezultate, obinute n urma optimizrii condiiilor de reacie,

vor fi mai apoi comparate cu cele rezultate n urma metabolizrii offline, dar i cu cele
rezultate n urma folosirii microzomilor umani. n final, vom testa versatilitatea i eficiena
sistemului nostru, prin monitorizarea inhibiiei CYP1A1, cu ajutorul apigeninei.
Primul pas n dezvoltarea unei proceduri EMMA este acela de a desfura o
serie de teste preliminare, n ncercarea de a defini principalii factori ce influeneaz
metabolizarea. n cazul acestui studiu, au fost realizate dou serii de experimente
preliminare, principala diferen dintre cele dou fiind modul de introducere al probei n
capilar (injectare la anod versus injectare la catod).
Pentru nceput, n cadrul injectrii la anod, s-a elaborat o metod electroforetic
simpl pentru separarea substratului i a produsului de reacie. Este foarte important de
menionat, c la pH fiziologic, nici etoxicumarina, nici hidroxicumarina nu posed
mobilitate electroforetic proprie, migrnd n acelai timp cu fluxul electro-osmotic. Ca
i consecin, a fost folosit cromatografia electrocinetic micelar, dodecil-sulfatul de
sodiu devenind surfactantul de elecie, la o concentraie de 10 mM. Dup cum putem
observa n Figura II.3.1. , n separarea celor dou standarde au fost obinute o bun
rezoluie (RS=16) i un timp scurt de analiz.

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 14

EC
12

10

8
Absorbance

4
HC

-2
0 2 4 6 8 10

Time(min)

Tampon de separare: Tampon fosfat 50 mM la pH 7.4 + 10 mM DSS; Injectare la anod 50 mbar pentru 3
sec; Capilar netratat: 50 m d.i. i 48,5cm lungime total (40 cm lungime efectiv); Voltaj: 25 kV;
Temperatur: 37C; Detecie: 320 nm.
"

Figura II.3.1. Electroferograma obinut pentru separarea standardelor de HC i EC

Ulterior, un procedeu de analiz offline, urmat de unul online, au demonstrat

necesitatea optimizrii metodei, ntruct picul de hidroxicumarin a fost greu detectabil. n
acest stadiu, s-a ncercat i folosirea unui standard intern, atenololul i cetirizina fiind alei ca
reprezentani. Cu ajutorul cetirizinei, s-a demonstrat c standardul intern nu este metabolizat
n timpul experimentului, n timp ce cofactorul este metabolizat numai n prezena enzimei.
Totodat, stabilitatea n timp a reactanilor a fost confirmat (Figura II.3.6.).
n plus, n cadrul analizei prin electroforez capilar, se cunoate importana
ordinii de introducere a probelor n interiorul capilarei [45]. ntr-adevr, s-a observat o
diferen n cazul injectrii microzomi + substrat , cu o arie normalizat de 0.026 (n=3),
n timp ce pentru ordinea de injectare substrat + microzomi, aria normalizat a fost de
0.068 (n=3). Mai mult, concentraia microzomilor s-a dovedit a avea o influen major
asupra ratei metabolizrii.

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 25

20

Absorbance Cetirizin 0.05 mg/mL

15
NADPH 0.4 mg/mL

EC 0.1 mM
HC 0.1 mM
10

Microzomi 0.1 mg/mL

5 Tampon de separare

0
0 2 4 6 8 10

Fire 2.8. Test deTime(min)

stabilitate offline
Tampon de separare 50 mM la pH 7.4 + 10 mM DSS; Timp de ateptare: 30 min; Injectare la anod 50
mbar pentru 3 sec; Capilar netratat: 50 m d.i. i 48,5cm lungime total (40 cm lungime efectiv);
Voltaj: 25 kV; Temperatura: 37C; Detecie: 235 nm.
"
Figura II.3.6. Control de stabilitate offline

Dei n cadrul injectrii la anod s-a obinut un timp foarte scurt de migrare
pentru hidroxicumarin (2.40 minute), acest parametru ar putea fi optimizat prin
utilizarea injectrii la catod. n plus, timpul total al analizei va descrete, fr a necesita
procedee complicate de manipulare a probelor, metabolizarea avnd loc online.
De menionat, c a fost folosit un alt standard intern n cadrul acestei serii de
studii preliminare, i anume oxidul de mesitil 0.005%, vizibil n lumina UV la 235 nm.
Acesta posed un timp de migrare mult mai scurt dect cetirizina: 0.37 minute pentru
oxidul de mesitil, versus 11.91 minute pentru cetirizin.
Factorii cheie urmrii n continuare au fost: ordinea de injectare a probelor,
timpul de incubare, concentraia microzomilor i switch-ul de voltaj i durata acestuia.
Dintre modelele de introducere a probelor n capilar, cel care a generat cea mai
mare cantitate de hidroxicumarina a fost cel denumit modelul sandwich. Astfel, un
plug de enzim (microzomi), urmat de unul de substrat + cofactor + standard intern,
urmat la final de unul de microzomi, au fost supuse unui switch de voltaj dup cum
urmeaz : (+0,5 kV pentru 7s), (-0,5 kV pentru 7 s), (+0,5 kV pentru 7s), (-0,5 kV
pentru 7s). Conform Tabelului II.3.1., decizia de a utiliza aceast metodologie de
injectare pentru urmtoarele experimente, este justificat.

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Tabelul II.3.1. Impactul modelelor de injectare asupra ariei normalizate (NA) a HC

Aria TM NA HC Aria TM NA EC
HC (min) (CV) EC (min) (CV)
0.046 1.96
S+M (-Vs) 2.31 0.64 (6.3) 155.2 1.02 (8.5)
0.04 1.55
S+M (+Vs) 2.46 0.64 (11.0) 154.8 1.03 (9.2)
0.05 1.85
M+S (-Vs) 1.87 0.59 (5.1) 92.4 0.79 (5.2)
0.04 1.85
M+S (+Vs) 2.13 0.61 (11.5) 127.1 0.86 (5.8)
0.124 2.02
M+S+M ( -Vs) 4.63 0.62 (15.9) 97.6 0.81 (11.8)
0.132 2.08
M+S+M (+Vs) 5.56 0.55 (4.07) 112.4 0.71 (3.7)

Ulterior aplicrii acestui switch, este necesar un timp de repaus, cunoscut ca i

timp de incubare, pentru ca reacia s aib loc. Dei se obine o cantitate maxim de
produs de reacie dup 30 de minute, valoarea acestui parametru a fost setat la 10
minute, pentru a nu crete excesiv durata analizei.
nc o dat, a fost investigat influena concentraiei enzimei asupra cantitii de
hidroxicumarin. S-a observat c o concentraie a microzomilor de 4 mg/mL duce la
atingerea platoului n ceea ce privete rata de conversie a etoxicumarinei. Pentru a
rmne n zona liniar a curbei, dar i pentru a nu suprancrca capilara, concentraia
microzomilor a fost setat la 2 mg/mL.
La finalul acestei serii de studii preliminare, s-a testat i influena switch-ului de
voltaj i a duratei acestuia asupra metabolizrii. ntr-o prim instan, datele sugereaz
c pentru un numr constant de schimbri rapide de polaritate, aplicarea de poteniale
mai nalte duce la scderea cantitii de produs de reacie. O posibil explicaie este
aceea c reactanii nu interacioneaz suficient, etoxicumarina migrnd mai departe
dect enzima, avnd mobiliti electroforetice diferite (Tabelul II.3.2.). Probabil, timpul
pe care l petrec n contact cele dou entiti scade, o dat cu creterea voltajului.
Aceast ipotez este confirmat privind Tabelul II.3.3. Creterea duratei switch-
ului de voltaj atrage dup sine o descretere a ariei normalizate a hidroxicumarinei.
n plus, devine evident c cele doua variabile sunt interconectate i ar trebui
privite ca una singur, influena lor asupra procesului fiind de necontestat.

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Tabelul II.3.2. Influena switch-ului de voltaj asupra ariei normalizate (NA) a HC

Aria TM(min) NA HC Aria EC TM(min) NA EC

HC (CV) (CV)
0.5 kV 5.56 0.55 0.13 112.4 0.71 2.08
(4.1) (3.7)
1 kV 4.34 0.57 0.07 112.6 0.73 1.43
(7.0) (2.9)
2.5 kV 3.93 0.54 0.07 106.2 0.68 1.58
(7.5) (2.6)
5 kV 5.08 0.57 0.08 118.9 0.74 1.43
(5.6) (2.3)

Tabelul II.3.3.Influena duratei switch-ului de voltaj asupra ariei normalizate (NA) a HC

Aria TM(min) NA HC Aria EC TM(min) NA EC

HC (CV) (CV)
2 sec 4.19 0.56 0.065 113.97 0.72 1.37
(8.1) (7.4)
6 sec 4.67 0.54 0.093 112.74 0.69 1.77
(4.8) (6.5)
10 sec 5.10 0.59 0.075 122.61 0.79 1.36
(6.2) (6.4)

Ultima parte a optimizrii s-a axat pe studierea n amnunt a factorilor

identificai mai sus ca fiind primordiali. Principala diferen fa de studiile preliminare
este utilizarea de aceast dat a superzomilor, ce exprim preferenial o singur enzim
i anume, CYP1A1.
S-a nceput prin pstrarea aceluiai protocol de injectare a probei: un plug de
enzim (superzomii CYP1A1), un plug coninnd substratul i cofactorul ( EC i
NADPH) i din nou, la final, un plug de enzim. nainte i dup acestea, au fost
injectate alte dou plug-uri de tampon de separare ce nu conineau surfactant, pentru a
proteja enzima de denaturare.
Au fost efectuate patru schimbri rapide de polaritate, overlap-ul reactanilor
fiind obinut datorita diferenei de mobilitate electroforetic a acestora. Aa cum am
amintit mai sus, la pH-ul fiziologic de 7.4, EC este o molecul neutr, migrnd
mpreun cu fluxul electroosmotic. Pentru a ne asigura ca metabolizarea are loc, a fost
setat un timp de incubare de 15 minute. Totodat, a fost investigat temperatura
capilarei, prin comparaie ntre valorile de 25C si 37C. Nu a fost identificat nicio
diferen semnificativ, cu excepia unei valori mai ridicate a coeficientului de variaie

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pentru temperatura de 37 C ( 14.3 vs. 3.1). Este posibil ca, la temperaturi mai ridicate,
stabilitatea enzimei s fie afectat.
n continuare, s-au avut n vedere influena lungimii plug-urilor injectate asupra
ratei de metabolizare. Cele mai bune rezulate au fost obinute pentru o injectare de 6
secunde (Figura II.3.14.). Pentru valorile de 10, respectiv 2 secunde, fie forma picului a
fost afectat, fie cantitatea de HC a sczut considerabil.

EC

60

50
Absorbance (mAU)

40

30 NADPH

20 HC

C
10
B
A
0
0,0 0,5 1,0 1,5 2,0 2,5 3,0

Time (min)

Tampon de separare: Tampon fosfat 50 mM la pH 7.4 + 18 mM DSS; Capilar netratat: 50 m d.i. i

48,5cm lungime total (8.5 cm lungime efectiv); Mod sandwich de injectare; Temperatur:
25C; Switch de voltaj: +0,5 kV/-0,5 kV/+0,5 kV/-0,5 kV fiecare pentru 7s; Timp de
ateptare: 15 min; Voltaj: - 25 kV; Detecie: 320 nm.

Figura II.3.14. Influena lungimii plug-urilor asupra metabolizrii EC

Ulterior, s-a trecut la optimizarea parametrilor enzimatici, primii luai n calcul

fiind timpul de incubare i concentraia superzomilor. Cel mai bun compromis ntre un
timp de analiz total scurt i un turnover considerabil al substratului a fost atins prin
alegerea valorilor de 15 minute pentru timpul de incubare i o concentraie de 200
pmol/mL pentru superzomii CYP1A1.
Ultimul pas al optimizrii a fost reprezentat de conceperea unui design
experimental. Au fost definite ca i variabile doi dintre factorii cheie ai metabolizrii:
valoarea voltajului de mixing al plug-urilor (X1 ; trei nivele: 0.1, 0.55 i 1 kV) i durata

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aplicrii acestuia (X2; trei nivele: 2, 6 i 10 secunde). Aria corectat a HC a fost propus
ca variabil de optimizat, Y. Ecuaia care a definit relaia dintre cele trei:
Y= 0 + 1X1 + 2X2+ 12X1X2+ 11X11+ 22X22 + ,

unde 0 este interceptul, 1 i 2 sunt termenii efectului principal, 12 este termenul de

interaciune, 11 i 22 sunt termenii ptratici, iar este eroarea.
Pentru o mai bun vizualizare a rezultatelor, a fost conceput o diagram 3D
(Figura II.3.17.). Valoarea optim pentru voltajul de mixing s-a dovedit a fi 0.22 kV, n
timp ce durata aplicrii acestuia a fost stabilit la 10 secunde, dei graficul indic valori
superioare. Aceast decizie a fost luat n urma considerentului pstrrii unui timp de
analiz ct mai scurt.

Figura II.3.17. Suprafaa de rspuns a efectului voltajului de mixing i a duratei acestuia

asupra ariei corectate a HC

n urma setrii acestor condiii i a realizrii experimentului n triplicat, a fost

atins o valoare de 16.9 0.4 pentru aria corectat a HC (Figura II.3.18.). Calitatea
modelului predictiv al design-ului experimental a fost subliniat de faptul c valoarea
experimental se ncadreaz n intervalul de ncredere (17.2 0.7). n plus, deviaia
relativ standard, pentru toi parametrii urmrii, este mai mic de 2.1 %, valoare ce
poate fi considerat excelent dac inem cont de complexitatea sistemului.

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 Folosind o curb de calibrare standard, ariile picurilor de HC au fost
transformate n concentraii, cu scopul de a calcula rata reaciei metabolice. Cu valoarea
obinut de 67.8 1.4 M HC , pentru un timp de incubare de 15 minute i o
concentraie a enzimei de 200 pmol/mL, rata reaciei metabolice n condiii optime este
de 11.3 nmoli HC/min/nmoli de CYP1A1.

EC
50

40
Absorbance (mAU)

30 NADPH

20 HC

OFFLINE
10 MC EMMA

CYP1A1 online

0
0,0 0,5 1,0 1,5 2,0 2,5 3,0

Time (min)

Tampon de separare: tampon fosfat 50 mM la pH 7.4 + 18 mM DSS; Capilar neacoperit: 50 m d.i.

i 48,5cm lungime total (8.5 cm lungime efectiv); Temperatur: 25 C; Switch de voltaj : +0,22
kV/-0,22 kV/+0,22 kV/-0,22 kV fiecare pentru 10s; Timp de ateptare: 15 min; Voltaj: - 25 kV;
Detecie: 320 nm.

Figura II.3.18. CYP1A1 online vs. MC EMMA vs. Metabolizare offline

n completare, pentru a testa eficiena sistemului, s-a realizat o comparaie cu

procedura clasic a metabolizrii offline. Toate celelalte condiii experimentale au fost
meninute constante (Figura II.3.18.). Se poate observa c valoarea ariei corectate
pentru HC n condiii clasice (20.0 0.1) este cu puin mai ridicat dect cea obinut
online (16.9 0.4). Acest fapt se poate datora mixing-ului electroforetic, care nu este
total. Totui, metoda de fa este una complet automatizat, ce economisete timp i
reactani, spre deosebire de cea offline.
Aplicabilitatea sistemului prezentat este dovedit i de utilitatea sa n cadrul unor
poteniale studii in vitro. A fost efectuat un test, utiliznd microzomi hepatici

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metilcolantren-indui (2mg/mL), rezultatele fiind comparabile cu cele obinute inline
(Figura II.3.18.). Din nou, a fost confirmat selectivitatea metodei.
Un alt domeniu de aplicare al procedurii dezvoltate este cel al inhibiiei
enzimatice. Apigenina a fost definit ca i un inhibitor puternic al acitivitii CYP1A1.
Din Figura II.3.19. se poate observa o reducere semnificativ a ariei picului HC, n
urma incubrii enzimei cu 100 M apigenin, pentru 30 de minute.

EC

40

NADPH
Absorbance (mAU)

30

Prod. degrad.
apigenin

20

HC Cu apigenin
10

Fr apigenin

0
0,0 0,5 1,0 1,5 2,0 2,5 3,0

Time (min)

Tampon de separare: tampon fosfat 50 mM la pH 7.4 + 18 mM DSS; Capilar netratat: 50 m d.i. i 48,5cm
lungime total (8.5 cm lungime efectiv); Temperatur: 25 C; Switch de voltaj : +0,22 kV/-0,22 kV/+0,22 kV/-
0,22 kV fiecare pentru 10s; Timp de ateptare: 15 min; Voltaj: - 25 kV; Detecie: 320 nm.

Figura II.3.19. Apigenina ca i inhibitor CYP1A1

n concluzie, prezentul studiu descrie dezvoltarea i optimizarea unei metode

online complet automatizate de monitorizare a activitii CYP1A1. Separarea
compuilor de interes (7-hidroxicumarina, 7-etoxicumarina i NADPH) s-a realizat n
mai puin de 2 minute. Factorii cheie au fost identificai, studiai, design-ul experimental
demonstrnd importana investigrii fiecrui parametru n parte. n plus, consumul de
reactani este redus la minim.
Mai mult, rezultatele procedurii inline sunt comparabile cu cele ale
metabolizrii clasice offline. Totodat, metoda s-a dovedit a fi util i n cadrul studiilor

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de inhibiie enzimatic. Important de notat, aceasta este compatibil cu o varietate larg
de molecule, datorit utilizrii unui surfactant.
Avantajele evidente ale metodei optimizate sunt miniaturizarea i automatizarea,
ntruct reacia de biotransformare, separarea compuilor i detecia acestora are loc n
interiorul aceleiai capilare.

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 I. GENERAL PART

I.1. INTRODUCTION

Developing a new drug from innovative idea to the launch of a final product is a
complex investigation. Failure is frequently encountered if the new chemical entity
(NCE) lacks efficiency or safety [1]. For this reason, preclinical stage of this process has a
great importance, consisting of different steps : initial target identification and validation,
assay development, high throughput screening, hit identification, lead optimization and
selection of a candidate molecule for clinical development (Figure I.1.1.).

T\Target ID Candidate NDA

IND
& Selection" selection filing
filing

Basic Lead Preclinical Clinical FDA

Research Discovery Development Development Filing
"
Years 3 1 6 1.5

Figure I.1.1. Drug discovery process [1]

"

Since the toxicity and efficacy of drugs are closely related to their
pharmacokinetic properties, a better understanding of their metabolic pathways during
the hit to lead optimization stage is essential [2]. In this context, in vitro methods for
drug metabolism studies became mandatory. Firstly, they can be used in the early
preclinical stages and secondly, it is possible to use cells and liver fractions, or even
human enzymes, making the data more relevant for the human in vivo studies [3].
Moreover, for a better understanding and a good quantification of all the
processes involved, a potent analytical methodology needs to be employed [2].
Nowadays, there are multiple techniques available. Among them, capillary
electrophoresis offers fast analysis, with very high peak efficiencies, while respecting
the critical trends of bioanalytical assays, automation and miniaturization.

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I.2. METABOLIZATION STUDIES

I.2.1. Drug metabolism

Nowadays, the study of pharmacokinetics is a growing science. The
investigation of absorption, distribution, metabolism and excretion (ADME) of a new
chemical entity became mandatory. Drug metabolism is the process of conversion of a
lipophilic compound to a more hydrophilic one. The metabolites resulted are easily
eliminated from the human body [3].

Figure I.2.1. Drug metabolism [4]

"

In general, drug metabolism can be divided into Phase I and Phase II (Figure
I.2.1.). Phase I involves reduction, oxidation and hydrolysis reactions, mediated mainly
by cytochromes P450 (CYPs) and to a smaller extent by flavin-containing
monooxygenases (FMOs) [4]. Phase II metabolic enzymes, as UDP-
glucuronyltransferases or sulfotransferases, catalyze conjugation reactions. The
lipophilic chemicals react, for example, with uridine diphosphate-glucuronic acid, an
endogenous co-factor [3]. This leads to an increased solubility of drug metabolite in
water, making them more conveniently excreted from the organism [4].
However, CYPs are the dominant enzyme systems that control drug metabolism
and clearance, accounting 75% of the biotransformation of marketed pharmaceuticals.
They belong to a superfamily of heme-containing proteins, that catalyze the transfer of
an oxygen atom onto a xenobiotic or an endogenous compound. For example: steroids,

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fatty acids or prostaglandins can serve as substrate [5]. At the same time, they transport
electrons from nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) to
adrenodoxin, inside the microsome [6]. Due to the content in human liver and high
contribution to drug metabolism, only a few isoforms of CYP are of great importance
for most drug metabolism assays.
The most studied ones are: 1A family, 2A6, 2B6, 2C family, 2D6, 2E1 and 3A family
[3]. It is of great importance to notice that genetic polymorphism of some of these isoforms,
e.g., CYP2D6, CYP2C9 or CYP2C19, along with factors like gender, age or nutritional
habits, are at the base of the observed variability in CYP450-mediated drug clearance [5].

I.2.2. CYP 1 family

In the past few years, a new CYP family presented interest for the scientific world,
regarding its polymorphism and regulation activity. CYP 1 family consists of CYP1A1,
CYP1A2, and CYP1B1. In the human body, CYP 1A1 is found mainly in extrahepatic
tissues, as the uterus, thymus, pancreas or small intestine, with a great role in the
metabolism of xenobiotics [6]. It is also known as one of the most important enzymes in
the bioactivation of procarcinogens, leading to the obtention of reactive metabolites [7].
Also, CYP 1B1 is expressed in extrahepatic tissues such as the uterus, breast or prostate.
Moreover, it is overexpressed in tumor tissue [8].

Figure I.2.2. Mechanism of activation of CYP450 1 family

Their transcription is regulated through aryl hydrocarbon receptor, who is acting

as a ligand-activated transcription factor (Figure I.2.2.). A series of xenobiotics acts as
binding ligands: halogenated aromatic hydrocarbons, benzo()pyrene and polycyclic
aromatic hydrocarbons [6]. This generates the increase of transcription, CYP 1A1

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mRNA levels being very high in the lung cells of smokers, comparing with non-
smokers [7]. It is overexpressed also in breast cancer, liver, bladder, and colon [9,10].

Consequently, CYP1A1 in now considered an interesting target with respect to

cancer therapy.

I.2.3. In vitro metabolism studies

In order to be able to detect which enzyme is directly involved in the
metabolization process, the in vitro approach uses different models, working with:
fresh and cryopreserved hepatocytes, tissue slices, human liver microsomes [3] or
recombinant expressed enzymes (supersomes) [11].
Tissue slices, along with fresh or cryopreserved hepatocytes, are the most
complex in vitro model, reuniting phase I and phase II metabolic enzymes and co-
factors . If the first ones are really useful in the study formation of metabolites, the
second ones can estimate and predict the clearance and drug-drug interactions [3].
Human liver microsomes provide a more convenient way to study CYP-
mediated metabolism. They represent a subcellular fraction of tissue, which was
obtained by differential high-speed centrifugation [12], coming from single or multiple
individuals [11]. As a result, all of CYP enzymes are to be found in the microsomal
fraction, but not phase II enzymes [13]. An NADPH-regenerating system must be added
to this system, in order to preserve the co-factor function. By comparison with liver
slices, they are more rapidly available, although the last ones give the most closely
model to the in vivo situation [12]. Commercial microsomes can be purchased from
different companies, but a significant variability of their activity has been noticed from
vendor to vendor or batch to batch [14]. Therefore, it is recommended to check the quality of
the purchased batches. A simple method to verify their conformity is to run a UV
spectrum for carbon monoxide binding difference. The starting degradation of the
product is demonstrated by the appearance of a peak at 420 nm, and not at 450 nm [15].
In order to prevent a decrease in the enzymatic activity, human liver microsomes
should be stored at -80C [16]. If they are thawed and maintained on ice for less than 2
hours, they can be re-frozen and re-used. If not, their enzymatic activity is lost, the
major disadvantage being that after 2 hours of incubation at 37 C, the obtained results
would not be representative [15].
Supersomes, named also enzyme-only systems, allow the study of a single
metabolic enzyme. As in the case of human liver microsomes, the specificity is increased,

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this time, at a maximum level. If at the begging, they were obtained from recombinant
proteins in insect cells [11], molecular biology techniques allow nowadays the
successful cloning and expression of a large number of human CYP isoenzymes,
commercially available [3].
Another important characteristic of microsomes and supersomes is their
compatibility with modern analyzing techniques. There are two types of approaches:
off-line biocatalytical systems or on-line biocatalytical systems [4]. The last ones are
performed in flow-through system. In this manner, the reaction is directly coupled with
the separation and the detection of reactants. Techniques like liquid chromatography
(LC) or capillary electrophoresis (CE) are mandatory for this purpose.

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I.3. CAPILLARY ELECTROPHORESIS

I.3.1. Introduction
In 1974, Virtanen (Helsinki University of Technology, Finland) provided the
scientific world an early demonstration of the advantages of CE. The author described a
potentiometric detection method for zonal capillary electrophoresis, applied to the
quantitative analysis of the alkali cations Li+, Na+ or K+. The application area grew
considerably, including nowadays ionic or non-ionic compounds, organic or inorganic,
regardless of their molecular weight [17]. This physical method of analysis is based on
the migration, inside a capillary and under the influence of a direct-current electric field,
of charged analytes dissolved in an electrolyte solution [18].
Nowadays, CE has interesting applications in pharmacology-related assays,
enabling the study of proteins and proteins interaction. They can be analyzed even in
their native form, in physiological conditions [2]. Because this technique allows
miniaturization, CE-based enzyme assays are usually employed for enzyme kinetics or
enzyme inhibition studies [20]. It is also a great alternative to LC, offering unique
advantages: short time of analysis, low reagent consumption, minimal sample
requirement [19], high efficiency and ability to use different detection methods [21].

I.3.2. In-capillary assays

Initially, CE was employed only as a separation tool in enzyme assays.
Typically, the enzyme-catalyzed reactions were performed off-line, the incubation of
substrate, cofactor and enzyme being performed out of the capillary, in a separate vial.
However, there are some limitations. First of all, the time of the reaction and the time of
the analysis are different, a time delay being created. Secondly, the enzymatic reaction
must be stopped, by adding a new reagent to the solution. In addition, to avoid capillary
clogging or protein adsorption onto the capillary wall, a step of sample deproteinization,
prior to sample injection must be performed. For this purpose, centrifugation and
precipitation of the proteins are mandatory. Moreover, even though CE analysis requires
only a nanoliter scale sample, much larger amounts of materials are used [19, 22, 23].
More recently, CE was employed as an on-line micro reactor. In this kind of
assays, the capillary has multiple functions; it serves as a mini reactor, as a separation
medium for both reaction products and resulted products and as a detection cell.

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 I.3.3. Electrophoretically mediated microanalysis

I.3.3.1. Theoretical background

Two methodologies are available to study on-line drug metabolism: the
heterogeneous mode and the homogeneous mode [4]. The first one contains one
immobilized reactant, most often the enzyme, on the capillary wall, while the others
components are present in the liquid phase or background electrolyte (BGE). On the
contrary, in homogeneous assays, all reactants are present in solution [23].
Bao and Regnier [24] described the first in-line capillary electrophoretic
enzymatic assays. In their study, a plug of the enzyme (glucose-6-phosphate
dehydrogenase) was injected into a capillary previously filled with the substrate,
glucose-6-phosphate. The cofactor, nicotinamide adenine dinucleotide phosphate
(NADP) was also present in the capillary. Finally, under the influence of an electric
field, the formed product NADPH, was detected at 340 nm. This type of assay gained
the name of electrophoretically mediated microanalysis or EMMA. It is important to
notice that the enzyme and the substrate injected had different electrophoretic
mobilities, which made possible their electrophoretic mixing, their separation, and
quantification. By all means, EMMA defines herself as a homogeneous enzyme assay.
This approach offers automation, miniaturization and eliminates samples handling.

I.3.3.2. EMMA modes

In general, two major EMMA formats are used to load and mix reagents in a
capillary, namely continuous engagement EMMA or long contact mode and transient
engagement EMMA, known also as the plug-plug format. For the first type, one of the
reactants completely fills the capillary, whereas a plug of the other reactant is
introduced during analysis (zonal sample introduction, Figure I.3.1.A.). The second
reactant can also be continuously injected from the inlet vial (moving boundary sample
introduction) [23]. Product formation begins under the influence of a voltage mixing.
It is important to emphasize that in the inlet vial is maintained the faster
migrating reactant, while the slower migrating one is in the capillary. In this manner, an
overlap of the two chemical species can be performed, because they interpenetrate
inside of the formed micro-reactor [22].

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 Figure I.3.1. EMMA modes [25]
The plug-plug mode (or short contact mode, Figure I.3.1.B.) consists of the
introduction of the enzyme and substrate as different plugs, the one with a lower
electrophoretic mobility being injected first. The enzymatic reaction takes place also at the
application of an electric field when the zones overlap. As a result, the product(s) and the
unreacted substrates are electrophoretically transported toward the detector. There is one
issue with this methodology because the electrophoretic conditions (as the composition and
pH of the BGE) must be favorable for both the enzymatic reaction and the separation itself.
If not, the enzyme may lose its activity when in contact with the buffer [19, 23].
To overcome this limitation, Van Dyck and coworkers [26] introduced the
partial filling technique (Figure I.3.1.C.). In this setup, the capillary has two fillings: a
buffer for the enzymatic reaction and one for the separation and migration of the
resulted products.
Moreover, there are three ways of mixing the reagent plugs. Firstly, if we deal
with an enzyme that is not resistant at an electric field, the reaction takes place under no
applied voltage. The reactants are injected in a sandwich plug mode (substrate-enzyme-
substrate) and their mixing is realized by longitudinal diffusion (Figure I.3.1.D.). But,

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when working with multiple plugs, this method seems to be not efficient. Hereby,
mixing by transverse diffusion of laminar flow profiles (TDLFP) has been proposed
(Figure I.3.1.E.) [27, 28]. Using pressure, a series of consecutive plugs is introduced
into the capillary. Due to the high force of injection and to the laminar flow formed
inside the capillary, the nondiffused plugs will interfere with the previously injected
ones. After injection, the plugs are not mixed by longitudinal diffusion. Is the transverse
diffusion that allows the reaction to take place.
In addition, to further increase the efficiency of in-capillary EMMA assays,
Sanders et al. [29, 30] introduced a procedure based on the plug-plug mode. The
innovation consists in the use of a successive mixing, by rapid polarity switches (RPSs).
Post-injection, a series of positive and negative potentials are applied. This backward
and forward movements equals to a mechanical shaking, permitting the reaction to
initiate (Figure I.3.1.F.). RPS provides more reproducible product peaks and can even
help to an accurate determination of molecules of interest. To gain all these advantages,
attention should be paid to the size of the injected plug and to the rate of the
electroosmotic flow.

I.3.3.3. Development and optimization

The development of an EMMA method is challenging, due to the fact that it
implies optimized procedures for both the enzymatic reaction and the analytes
separation. Although there are a series of papers describing EMMA enzymatic assays,
little work has been done on the study and optimization of the specific parameters of an
in-line reaction [2].

I.3.3.3.1. Key factors

Experimental factors, such as enzyme concentration, mixing voltage and mixing
voltage time, should be considered when perfecting an EMMA method. Stahl et al. [31]
proved the importance of a design of experiment, investigating the effects of
conductivity on zone overlap, by computer simulation and by experiment. The influence
of each factor is unique and really important to study, the authors describing both
general and particular trends for the migration of molecules.
Furthermore, when working with proteins in CE, an important issue to consider
is their adsorption to the capillary wall. The major resulting problem is the clogging of
the capillary [23]. Luckily, artificial microsomes, that include recombinant DMEs (drug

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metabolizing enzyme), are not containing a high amount of protein, as purified
preparations of natural hepatic cells. The disturbance of results can be avoided with a
good rinsing step between runs. Yet, some authors have suggested other possible ways
to avoid alteration of the capillary. Pauwels et al. [32] used multi-walled carbon
nanotubes (MWNTs) to improve capillary lifetime. Large amounts of proteins or even
long incubation times are some criteria for the use of MWNTs. They do not only
prevented the interaction between silanol groups and proteins, but they also improved
the repeatability of peaks symmetry.
Another important factor to look after is the temperature of the electrolyte [23,
32]. If the conventional off-line assays are taking place at room temperature, 25C, in
the case of on-line systems, temperature choice is more complicated. Commonly,
enzymatic reactions should take place at physiological temperature, 37C. Sometimes,
in order to be fully productive, they may demand higher temperatures. Even if the
capillary is in contact with a thermostabilized heat exchanger, there are parts that are not
properly temperature-controlled, as the inlet. An ingenious solution has been proposed
for this problem [33], the authors using two different methods for temperature
measuring inside the capillary. The tray was also thermostated, in order to prevent the
exposure of the molecules to the elevated temperatures.

I.3.3.3.2. Injection, separation, and detection

The duration of the analysis in CE is given by the migration time of the analytes [34].
There are several approaches that can be used to increase the throughput of this technique:
- Reducing the length of the capillary;
- Increasing the separation voltage;
- Modifying the composition of the buffer;
- Dynamic coating of the capillary;
- Increasing the temperature.
The first option is the most commonly used, a capillary length of 25-30 cm being
the inferior limit at which an analysis can be performed. However, there is an
asymmetry between the position of the detector and the ends of the capillary. There is a
shorter part close to the outlet, of 7-10 cm, that can be used for different procedures.
Electrokinetic or hydrodynamic injection can be performed, electrophoresis being
realized at reverse polarity, using a sample placed at the outlet (Figure I.3.2.).

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 Figure I.3.2. Short end injection vs. normal injection [34]

All other necessary procedures remain intact, as for the normal injection mode.
This so-called short-end injection procedure can be integrated into EMMA [34, 35],
with the purpose of decreasing the total analysis time.
As for the separation method, the choice depends on the kinds of reactants and
products to be separated. As in other CE application fields, capillary zone
electrophoresis (CZE) is the most frequently used, followed by micellar electrokinetic
chromatography (MECK).
MECK [36, 37], as its name indicates, is based on the use of an ionic micellar
solution. By a partitioning mechanism, the analytes interact with the micelles, similar to
a chromatographic method. In this case, the mobile phase is represented by the EOF.
In order to create a pseudostationary phase from micelles, a surfactant is added
to the BGE. His concentration must be above its critical micellar concentration (CMC).

"

Figure I.3.3. Micellar Electrokinetic Capillary [36]

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 Sodium dodecyl sulfate (SDS), an anionic surfactant, is the most encountered
surfactant in this type of procedures. The principle of separation is explained in Figure
I.3.3. and can be defined as follows : The formed micelles are electrostatically attracted
towards the anode .The EOF transports the bulk solution towards the negative electrode
due to the negative charge on the internal surface of the silica capillaries. But the EOF is
usually stronger than the electrophoretic migration of the micelles and therefore the
micelles will migrate also toward the negative electrode with a retarded velocity[37].
This approach is really useful when a neutral compound has to be analyzed.
Finally, for the detection step, when working with MECK, some technical
aspects need to be taken into consideration. If the surfactant solution is transparent to
ultraviolet light, photometric detection can be used. But this type of detection has a low
sensitivity in terms of concentration. On the other hand, laser-induced fluorescence
(LIF) detection is very sensitive. It can work at a nanomolar scale while maitaining the
same precision. Moreover, the micelles can enhance this property. Another sensitive
detector is the electrochemical one. Recently, mass spectrometry (MS) became
mandatory when working with CE, especially on a nanoliter scale. Several interfaces for
CE-MS are available, but significant work has to be done for the development of
reproducible CE-MS [37].

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 II. EXPERIMENTAL PART

II.1. INTRODUCTION

In vitro studies for drug metabolism have a growing importance in the

characterization of new chemical entities. EMMA has proven to be an advantageous
technique for this type of studies, considering the high efficiency and short analysis
time that can be attained [38]. The capillary becomes a microreactor, where analytes
react, being also separated and detected, minimizing the sample consumption.
Using this approach, Curcio et al. described the activity of CYP450 using
dextromethorphan as substrate [5]. Zhang et al. [39] also performed an experiment for
the activity characterization of CYP3A4, testosterone, and nifedipine being tested as
substrates. In addition, the enantioselective metabolization of fluoxetine by CYP2D6
[40], the stereoselective N-demethylations of verapamil[41] and ketamine[42] were
investigated, proving the versatility of EMMA. In addition, some factors that influence
the electrophoretic process were tested, by univariate approaches, like the effects of
reagent zone and buffer conditions [43]. Another group [30] performed a computer
simulation with the purpose of predicting zone overlap, while others [28] described the
importance of rapid polarity switches.
In this paper, a fully automated in-capillary system for studying the activity of
CYP1A1 was developed [2]. 7-ethoxycoumarin (7-EC) was chosen as the substrate,
showing the best Km value for our enzyme (10 0.1M) [44]. In the presence of
nicotinamide adenine dinucleotide phosphate reduced (NADPH), as a cofactor, 7-EC
undergoes a reaction of O-deethylation, giving rise to 7-hydroxycoumarin (7-HC), the
molecule that will be assayed in our study. Optimization by multivariate approach was
employed, using a design of experiment (DoE). To the best of out knowledge, is for the
first time that a DoE is performed, in order to gain a better understanding of factors that
influence the metabolic reaction.
The best results obtained in optimal conditions were compared with:

- offline metabolization ;
- human liver microsomes.
Finally, we demonstrated the versatility of our method by monitoring CYP1A1
inhibition, using apigenin as a potent inhibitor.

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II.2. MATERIALS AND METHODS [2]

II.2.1. Instrumentation and capillaries

An HP 3D CE system (Agilent Technologies, Waldbronn, Germany) was used

for all the experiments. It was equipped with an autosampler, an oncolumn DAD and a
temperature control system. For instrument control, data acquisition and analysis, a
chemstation (Hewlett-Packard, Palo Alto, CA, USA) was employed.

Uncoated bared fused-silica capillaries were provided by ThermoSeparation

Products (San Jose, CA, USA). A total length of 48.5 cm ( 8.5 cm effective length) was
used, while the internal and external diameters were 50 and 375 m. The capillary was
thermostated at 37C by high-velocity air stream, whereas the external water bath was
kept at 25C, unless specified otherwise.

All the work was performed in the Laboratory for the Analysis of Medicines,
Universite de Liege, Belgium, within Erasmus+ Scholarship during July-September 2014.

Capillary preconditioning was realized daily, by flushing at 37C as follows:

- 1 M NaOH for 10 minutes;

- H2O for 10 minutes;
- BGE for 10 minutes.

Between runs, another rinsing procedure was followed:

- 1 M NaOH for 5 minutes;

- H2O for 5 minutes;
- BGE for 5 minutes.

To prevent carryover, the capillary ends were dipped into water, after each
injection step.

II.2.2. Reagents and chemicals

The electrolytes: sodium dihydrogen phosphate (NaH2PO4), disodium hydrogen

phosphate (Na2HPO4) and magnesium chloride (MgCl2), were purchased from Merck
(Darmstadt, Germany). Dimethyl sulfoxide (DMSO) and sodium hydroxide (NaOH)
were from BDH Prolabo Chemicals (Leuven, Belgium), whereas sodium chloride
(NaCl) was obtained from Acros Organics (NJ, USA).
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 The reactants used for the enzymatic procedure: 7-EC and 7-HC were acquired
from Alfa Aesar (Karlsruhe, Germany); NADPH was purchased from Panreac
AppliCHem (Darmstadt, Germany); Supersomes CYP1A1 (1 nmol/mL) were supplied
from Corning Supersomes (Woburn, MA, USA), while methylcholanthrene-induced
(MC-induced) rat liver microsomes (20mg/mL) were kindly provided by Advanced
Technology Corporation (Liege,Belgium).

The surfactant, SDS, was from Fischer Scientific (Loughborough, UK). The
enzymatic inhibitor, apigenin, was purchased from Extrasynthese (Genay, France). The
organic solvent, methanol, was provided by J.T. Baker Chemicals (Deventer, the
Netherlands). Ultrapure water was supplied by a Milli-Q equipment (Millipore,
Bedford, MA, USA).

All reagents and chemicals were of analytical grade.

II.2.3. Buffer and sample preparation

The incubation buffer (IB) was prepared by adding a solution of 50 mM

NaH2PO4 to a solution of 50 mM Na2HPO4, under continuous stirring, until pH 7.4. In
order to obtain the BGE, 18 mM of SDS were mixed with the IB.

Stock solutions of 10 mM EC and 10 mM HC were prepared in methanol, while

a stock solution of apigenin was prepared in DMSO. They were stored at 8 C, protected
from light. To reach the appropriate work concentrations, they were subsequently
diluted with IB. As for the cofactor, a solution of 5 mM of NADPH was prepared
freshly each day, in 0.05 M MgCl2.

The supersomes were kept at -80 C, thawed rapidly at 37 C, in a water bath and
then diluted with the IB to reach appropriate final concentrations. Also, they were stored
on ice until use.

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 II.2.4. EMMA procedure

The partial-filling technique, namely sandwich mode, was chosen for the
EMMA procedure. Different plugs of reactants were injected (outlet injection),
following the next protocol:

- IB plug ( -50 mbar for 10 s);

- CYP 1A1 supersomes plug ( -50 mbar for 6 s);
- NADPH and 7-EC plug ( -50 mbar for 6 s);
- CYP 1A1 supersomes plug ( -50 mbar for 6 s);
- IB plug ( - 50mbar for 10 s).

The reaction (Figure II.2.1.) was initiated by the application of a voltage switch
( -0.2/0.2/-0.2/0.2 kV, each for 10 s). The voltage was then turned off during 15
minutes, allowing the metabolic reaction to take place. The separation of the
components was performed at -25 kV, the detection being achieved at 320 nm.

7- Ethoxycoumarin (EC)

H3C O O O

CYP1A1 + NADPH, 37C

7- Hydroxycoumarin (HC)

HO O O

Figure II.2.1. Enzymatic reaction

The experimental design and the subsequent analysis were carried out using JMP
software version 10.0 (SAS Institute, Cary, NC, USA). Thirty experiments were
defined, repeating the central point five times, while the others were repeated three
times. The statistical significant threshold was established at p value < 0.05.

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"
 In order to find the reaction rate (nmoles of HC/min/nmoles of CYP1A1), the
following mathematical equation was used for the calculation of the volume of each plug:

!"!! ! !!
!= ,
!"#!

where V (m3) is the injected volume, P (Pa) is the applied pressure, d (m) is the
capillary internal diameter, t (s) is the duration of pressure applications, is the solution
viscosity and L (m) is the capillary length.

II.2.5. Offline incubation procedure

The metabolization took place in an Eppendorf placed in a water bath (Heidolph

Instruments, Schwabach, Germany), during 15 minutes at 37C. 10L of 1 pmol/L
supersomes were mixed with 20 L of IB, 10 L of 5mM NADPH and 10 L of 1.25
mM EC. After the given time, the reaction was stopped by addition of 10 L of a
mixture of 0.05 M NaOH/NaCl.

Using a centrifuge (Eppendorf, Hamburg, Germany), at a working speed of 13

400 rpm, for 5 minutes, the resulted supernatant was injected in our system. The outlet
injection was performed also in this assay, at -50mbar for 6 seconds. The separation was
carried out at -25 kV, the amount of HC being spectrophotometrically determined.

II.2.6. Inhibition assay procedure

The inhibitor, apigenin (100 M), was preincubated for 30 minutes with our
enzyme, 200 pmol/L CYP1A1 or 2 mg/mL MC-induced rat liver microsomes. The
exact protocol as for the EMMA procedure was consequently respected. The obtained
results were compared with the ones obtained after a blank EMMA procedure. Here, the
supersomes were preincubated with the solvent (DMSO 1% in BGE) employed for 100
M apigenin preparation.

17"
"
 II.3. RESULTS AND DISCUSSION

II.3.1. Preliminary studies - inlet injection optimization

II.3.1.1. Development of a CE method for EC and HC separation

The first step in the development of an automated EMMA procedure is to develop a

simple CE method for the substrate (EC) and product (HC) separation.

It is really important to notice that, at physiological pH, neither EC or HC dont

possess an electrophoretic mobility, migrating with the electro-osmotic flow (EOF). As
a result, MECK had to be used. For this purpose, a surfactant, namely SDS at a
concentration of 10 mM was used.

14

12

10

8
Absorbance

EC"

2 HC"

-2
0 2 4 6 8 10

Time(min)

BGE: Sodium phosphate buffer 50 mM at pH 7.4 + 10 mM SDS;Inlet injection 50 mbar for 3 sec;
Uncoated fused silica capillaries: 50 m i.d. and 48,5cm total length (40 cm effective length); Voltage: 25 kV; Capillary
temperature: 37 C;Detection: 320 nm.
"

Figure II.3.1. The electropherogram obtained for the separation of HC and EC

standards

18"
"
 As seen in the Figure II.3.1., a good resolution (RS =16) and a short analysis
time were obtained for the separation of the two standards. The same IB was maintained
for all the following experiments.

II.3.1.2. Offline metabolization

The next step was to investigate the behavior of our substrate in the presence of
the enzyme. For this reason, an ATC protocol was followed, performing an off-line
metabolization assay, using microsomes. After a waiting time of 15 minutes, the
reaction was stopped using a mixture NaOH/NaCl and subjected to a centrifugation
step. The resulted supernatant was injected in the CE, obtaining the adjacent
electropherogram (Figure II.3.2.).

4 EC"

3
Absorbance

2
NADPH"
1 HC"

-1
0 1 2 3 4 5 6 7

Time(min)

BGE: Sodium phosphate buffer 50 mM at pH 7.4 + 10 mM SDS; WT: 15 min; Inlet injection 50 mbar
for 3 sec; Uncoated fused silica capillaries: 50 m i.d. and 48,5cm total length (40 cm effective length);
Voltage: 25 kV; Capillary temperature: 37 C;Detection: 320 nm.
"
Figure II.3.2. The electropherogram obtained for offline metabolization preliminary
studies

19"
"
 As expected, the peak of HC emerged, along with the one of the cofactor. By
comparison with Figure II.3.1., the retention times were similar for substrate and
reaction product, proving that the choice of this reactants is justified. In addition, there
are no interferences at the chosen wavelength.

II.3.1.3. Online metabolization

To perform inline assays, the EMMA approach was selected, allowing the
enzymatic reaction to take place directly inside the capillary. A sequential injection
protocol was used, in order to reduce the manipulation steps. In a previously BGE-filled
capillary, 4 different plugs were injected at inlet: BGE without SDS, microsomes, EC
and NADPH in BGE and again, BGE without SDS and a voltage switch was applied.

12

EC"
10

NADPH"
Absorbance

HC"
2

-2
0 2 4 6 8 10 12

Time(min)

BGE: Sodium phosphate buffer 50 mM at pH 7.4 + 10 mM SDS; Uncoated fused silica capillaries: 50 m i.d.
and 48,5cm total length (40 cm effective length);Sequential injection : (50 mbar for 10s), (50 mbar for 6s), (50
mbar for 6s), (50 mbar for 10s);Voltage switch : (+0,5 kV for 7s), (-0,5 kV for 7 s), (+0,5 kV for 7s), (-0,5 kV
for 7s); Waiting time: 15 min; Migration voltage: 25 kV; Capillary temperature: 37 C; Detection: 320 nm.

Figure II.3.3. The electropherogram obtained for online metabolization preliminary

studies

20"
"
 The on-line metabolization gave rise to a smaller amount of metabolite (Figure
II.3.3.), emphasizing the need of optimization of various key parameters involved in the
metabolic reaction.

II.3.1.4. Use of internal standards

In order to decrease the variability of the CE analysis, the use of an internal

standard (IS) is justified. The first two tested were atenolol (Figure II.3.4. A.), a
selective 1 receptor antagonist or beta-blocker, and cetirizine (Figure II.3.4. B.), a new
generation drug of H1-antihistamines.

"

"

A.

B.

Figure II.3.4. Chemical structures of atenolol and cetirizine

Their behavior has been observed by an off-line procedure, after a waiting time
of 30 minutes. The major difference between this two assays and the previous ones is
the working wave length of 235 nm. If atenolol shows weak resolution (Figure
II.3.5.A.), cetirizine provides a better separation (Figure II.3.5.B.), but with a notably
increased migration time.

21"
"
 16

14 A.
12 Atenolol
10
Absorbance

0 2 4 6 8 10

Time (min)"

12

B.
10

Cetirizine
8
Absorbance

0
0 2 4 6 8 10 12 14

Time(min)

BGE: Sodium phosphate buffer 50 mM at pH 7.4 + 10 mM SDS; WT :30 min; Inlet injection 50 mbar for 3
sec; Uncoated fused silica capillaries: 50 m i.d. and 48,5cm total length (40 cm effective length); Voltage: 25
kV; Capillary temperature: 37 C; Detection: 235 nm.
"

Figure II.3.5. Atenolol (A.) and cetirizine (B.) as internal standards

22"
"
 Despite the prolonged time of analysis, cetirizine was further used in a series of
off-line control tests, in order to check the stability and possible interactions of our
compounds.

Firstly, all the components of the enzymatic reaction were separately injected,
after an off-line incubation. The electropherogram (Figure II.3.6.) offers the certainty
that all the molecules are stable during the analysis, having distinct migration times.

25

20

Cetirizine 0.05 mg/mL

Absorbance

15
NADPH 0.4 mg/mL

EC 0.1 mM
HC 0.1 mM
10

Microsomes 0.1 mg/mL

5 Neat buffer

0
0 2 4 6 8 10

Time(min)
BGE: Sodium phosphate buffer 50 mM at pH 7.4 + 10 mM SDS; WT: 30 min; Inlet injection 50 mbar for
3 sec; Uncoated fused silica capillaries: 50 m i.d. and 48,5cm total length (40 cm effective length);
Voltage: 25 kV; Capillary temperature: 37 C; Detection: 235 nm.
"

Figure II.3.6. Offline stability control test

Secondly, another control test was performed, this time only NADPH and
cetirizine being compared. As shown both in Figure II.3.6. and Figure II.3.7., cetirizine
is not metabolized, not even in the presence of microsomes. On the other hand, NADPH
is consumed and degraded, but only in the presence of the enzyme.Therefore, the use of
an internal standard proves to be a useful tool when monitoring the substrate.

23"
"
 25

20

Absorbance
15

NADPH NADPH NADPH CT"

10 related related

0
0 2 4 6 8 10

Time (min)"
BGE: Sodium phosphate buffer 50 mM at pH 7.4 + 10 mM SDS; WT: 15
NADPH+CT min; Inlet injection 50 mbar for 3 sec; Uncoated fused silica capillaries: 50
NADPH m i.d. and 48,5cm total length (40 cm effective length);
Voltage: 25 kV; Capillary temperature: 37 C; Detection: 235 nm.
"

Figure II.3.7. Off-line control test

II.3.1.5. Injection order

In a CE analysis, a particular aspect of the process is represented by the

introduction of the sample into the capillary [45]. The right approach for the right
application can lead to significant improvements in performance, particularly with
regard to sensitivity. Injection order seems to be crucial, especially when performing an
on-line reaction.

To demonstrate this principle, methylcholanthrene-induced microsomes (MC)

0.5 mg/mL were injected before and after the substrate, as different plugs. After a
waiting time of 10 minutes, with no voltage switch, the normalized areas (NA) obtained
of HC were compared. For the MC+substrate, NA was 0.026 (n=3), while for
substrate+MC, NA was 0.068 (n=3).

Taking into account that all the other experimental conditions were maintained
constant, it is clearly that injection order is a key factor for on-line CE metabolization.

24"
"
 II.3.1.6. Microsomes concentration

The single most important property of enzymes is the ability to increase the rates
of reactions occurring in living organisms, a property known as catalytic activity [46].
This activity is greatly influenced by pH, temperature, substrate concentration or
enzyme concentration. Normally, the reaction rate increases as the concentration of the
catalyst is increased.

In our case, at a concentration of MC 1 mg/mL versus a concentration of MC 0.1

mg/mL, a visible difference is spotted on the electropherogram (Figure II.3.8.). This
leads to an essential increase of MC concentration for the on-line metabolization.

16

14

12
EC"
10
Absorbance

6 MC"0.1"mg/mL"

4
EC"
HC"
2
MC"1"mg/mL"
0

-2
0 2 4 6 8 10

Time(min)

BGE: Sodium phosphate buffer 50 mM at pH 7.4 + 10 mM SDS; Inlet injection 50 mbar for 3 sec;
Uncoated fused silica capillaries: 50 m i.d. and 48,5cm total length (40 cm effective length); No voltage
switch; Waiting time: 10 min; Voltage: 25 kV; Capillary temperature: 37 C; Detection: 320 nm.
"

Figure II.3.8. Impact of the microsomes concentration

25"
"
 II.3.2. Preliminary studies - outlet injection optimization

Even though an inlet injection produced a very short time of CE analysis (2.40
min for HC peak), an outlet injection may permit decreasing, even more, this parameter.
Also, the total time of analysis is reduced, with no need for sample manipulation, in-line
metabolization being the chosen protocol.

Another series of preliminary studies was carried out by optimizing more key
factors, presented below. Some of them will be next included as variables in a design of
experiment.

Extremely important to mention that another IS was employed, namely mesityl

oxide (MO) 0.005%, visible in the ultraviolet light at 235 nm (Figure II.3.9.), since it
migrates faster than cetirizine: 0.37 minutes for MO versus 11.91 minutes for cetirizine.

25
HC

20 MO
EC
Ansorbance(mAU)

15

10

0,0 0,5 1,0 1,5 2,0 2,5 3,0

Time(min)

BGE: Sodium phosphate buffer 50 mM at pH 7.4 + 10 mM SDS; MO 0.005% ; Inlet injection -50 mbar for
3 sec; Uncoated fused silica capillaries: 50 m i.d. and 48,5cm total length (8.5 cm effective length); No
voltage switch; Migration voltage: -25 kV; Capillary temperature: 37 C; Detection: 235 nm.
"

Figure II.3.9. MO as internal standard

26"
"
 II.3.2.1. Injection order and voltage switch

As exposed before, injection order and mixing voltage switch are crucial
parameters for the obtainment of good reaction rates. Three alternatives were proposed
(Figure II.3.10.), applying or not a voltage switch of (+0,5 kV for 7s), (-0,5 kV for 7 s),
(+0,5 kV for 7s), (-0,5 kV for 7s).

?"
BGE MO+EC BGE
+" without
SDS
+NADPH Microsomes without
SDS
BGE pH 7.4 (+ 15mM SDS)
50 mM phosphate buffer

BGE MO+EC BGE

+" without
SDS
Microsomes +NADPH without
SDS
BGE pH 7.4 (+ 15mM SDS)
50 mM phosphate buffer ?"
" "

BGE MO+EC BGE

+" without
SDS
MC +NADPH MC without
SDS
BGE pH 7.4 (+ 15mM SDS)
50 mM phosphate buffer ?"
"
"

Figure II.3.10. Alternatives of outlet injection

The third approach, named also the sandwich mode, combined with rapid
polarity switches , generated the biggest amount of HC (Table II.3.1).

Tabel II.3.1. Outlet injection modes

Area TM NA HC Area TM NA EC
HC (min) (RDS) EC (min) (RDS)
0.046 1.96
S+M (-Vs) 2.31 0.64 (6.3) 155.2 1.02 (8.5)
0.04 1.55
S+M (+Vs) 2.46 0.64 (11.0) 154.8 1.03 (9.2)
0.05 1.85
M+S (-Vs) 1.87 0.59 (5.1) 92.4 0.79 (5.2)
0.04 1.85
M+S (+Vs) 2.13 0.61 (11.5) 127.1 0.86 (5.8)
0.124 2.02
M+S+M ( -Vs) 4.63 0.62 (15.9) 97.6 0.81 (11.8)
0.132 2.08
M+S+M (+Vs) 5.56 0.55 (4.07) 112.4 0.71 (3.7)

27"
"
 II.3.2.2. Incubation time

After performing the RPS, a limited period of time is necessary for the
enzymatic reaction to take place. As generally known, the incubation time must be
chosen with respect to the total time of analysis. In addition, this variable must permit
the reaction to reach a high conversion rate for the substrate.

In our case, a maximum quantity of HC is obtained after 30 minutes of

incubation (Figure II.3.11.). But in order not to increase excessively the analysis and to
remain in the linear part of the curve, the value of 10 minutes was chosen and set for all
the others further experiments.

II.3.2.3. Microsomes concentration

Once again, the impact of the enzyme concentration over the HC obtained
concentration was tested. It appears that at 4 mg/mL microsomes, the plateau is reached
and a further increase of the enzyme concentration produces no increase in the HC
amount (Figure II.3.12.).

In order to avoid capillary overload, but also to remain in the linear zone of the
curve, the microsomes concentration was set at 2 mg/mL.

35.00" 0.600"
30.00" 0.500"
25.00"
0.400"
C 20.00" N
A 15.00" A 0.300"
0.200"
10.00"
5.00" 0.100"

0.00" 0.000"
0" 20" 40" 60" 80" 0" 20" 40" 60" 80"
Incubation time(min) Incubation time(min)

BGE: Sodium phosphate buffer 50 mM at pH 7.4 + 12.5 mM SDS; Uncoated fused silica capillaries: 50 m i.d. and 48,5cm
total length (8.5 cm effective length); Sandwich mode injection; Voltage switch : +0,5 kV/-0,5 kV/+0,5 kV/-0,5 kV each for
7s; Migration voltage:- 25 kV; Capillary temperature: 37 C; Detection: 320 nm.

Figure II.3.11. Impact of incubation time on hydroxycoumarin CA and NA

28"
"
 25.00" 0.30"

20.00" 0.25"

0.20"
15.00"
C N 0.15"
A 10.00" A
0.10"

5.00" 0.05"

0.00"
0.00"
0" 1" 2" 3" 4" 5" 6"
0" 1" 2" 3" 4" 5" 6"

MC concentration (mg/mL) MC concentration (mg/mL)

BGE: Sodium phosphate buffer 50 mM at pH 7.4 + 12.5 mM SDS; Uncoated fused silica capillaries: 50 m i.d. and 48,5cm
total length (8.5 cm effective length); Sandwich mode injection; Voltage switch : +0,5 kV/-0,5 kV/+0,5 kV/-0,5 kV each for
7s; Wainting time: 10 min; Migration voltage:- 25 kV; Capillary temperature: 37 C; Detection: 320 nm.

Figure II.3.12. Impact of MC on hydroxycoumarin CA and NA

II.3.2.4. Voltage switch and switch time influence

In the attempt to investigate the most important variables interfering with our
experiment, voltage switch influence generated interesting results. At first glance (Table
II.3.2.), these data may appear to indicate that, for a constant number of RPS events,
higher potentials give rise to lower yield. This migth be due to the fact that our reactants
do not interact properly, EC migrating further than our enzyme, while having different
electrophoretic mobilities. Probably, the time they spend in contact decreases with the
increase of the voltage.

As a result, one might expect that at a higher switch time, the NA of HC would
decrease. Observing the data (Table II.3.3.), it becomes clear that this hypothesis is
valid. In addition, its certain that this two parameters should be considered as one, their
influence over the metabolization process being significant.

29"
"
 Table II.3.2. Voltage switch influence

Area TM(min) NA HC Area TM(min) NA EC

HC EC
0.5 kV 5.56 0.55 0.13 112.4 0.71 2.08
(4.1) (3.7)
1 kV 4.34 0.57 0.07 112.6 0.73 1.43
(7.0) (2.9)
2.5 kV 3.93 0.54 0.07 106.2 0.68 1.58
(7.5) (2.6)
5 kV 5.08 0.57 0.08 118.9 0.74 1.43
(5.6) (2.3)

Table II.3.3. Voltage switch time impact

Area TM(min) NA HC Area TM(min) NA EC

HC EC
2 sec 4.19 0.56 0.065 113.97 0.72 1.37
(8.1) (7.4)
6 sec 4.67 0.54 0.093 112.74 0.69 1.77
(4.8) (6.5)
10 sec 5.10 0.59 0.075 122.61 0.79 1.36
(6.2) (6.4)

II.3.3. Optimization- final steps [2]

As observed from the preliminary studies, some of the most important factors to
be taken into consideration are incubation time, injection mode, the concentration of the
enzyme and substrate, voltage switch and voltage switch time.

The main difference from the preliminary studies is the use of supersomes in the
process of metabolization. In this manner, we monitor the activity and behavior of a
single enzyme, CYP1A1. We started by keeping the same injection protocol, the
sandwich mode. Three distinct plugs of reactants were employed: first, the enzyme
(CYP 1A1 supersomes), secondly, the substrate and the cofactor ( EC and NADPH) and
again, the enzyme. Once again, before and after the CYP1A1 plugs, two plugs of BGE
without SDS were injected, to protect the supersomes from surfactant denaturation. The
hydrophobic tails of the SDS may disrupt the hydrophobic interactions in the interior of
the protein, which favorize protein denaturation.

30"
"
 II.3.3.1. Optimization of incapillary metabolization procedure

EMMA procedure is compatible with the use of supersomes and as seen above,
RPS was the method of choice for plug mixing.

Four rapid polarity switches were carried out, the overlap being obtained due to
te differences in the electrophoretic mobilities of the reactants. At pH of 7.4, EC is a
neutral molecule, migration with the EOF (ep=0 ; ap = EOF = (565.9 2.4) 10-6
cm2V-1s-1). On the other hand, the enzyme has its own electrophoretic mobility (ep= (-
162.7 2.8) 10-6 cm2V-1s-1, ap= 403.2 10-6 cm2V-1s-1).

The incubation time was settled at 15 minutes, even though the preliminary
studies showed a good conversion rate for 10 minutes. During this time, no voltage was
applied (zero potential amplification step). A constant voltage of -25 kV was applied
afterward for the separation step.

Also, the capillary temperature was investigated. No significant difference was

observed (Figure II.3.13.); the CA for HC at 25C was 14.5, while at 37C was found to
be 15.2. But it is noteworthy to mention that the RDSs obtained using 37C (14.3%)
were higher than those observed at 25C (3.1%), showing that at 37C, the stability of
the enzyme is probably affected.

Moreover, the final concentration of the organic solvent has to be kept as lower
as possible in the microreactor, in order to maintain the enzymatic activity. Methanol
was, in this case, limited at 2.5%, to conserve the supersomes function.

The next step was the study of the influence of injected plug length on the
metabolization rate. The best results were obtained by injecting at -50 mbar for 6
seconds each plug (Figure II.3.14.B.). With longer plugs (Figure II.3.14.C.), the shape
of the peak was affected, while with shorter plugs (Figure II.3.14.A.), the amount of HC
lowered.

31"
"
 40

30

Absorbance(mAU)

20

10 37 C

25 C

0
0,0 0,5 1,0 1,5 2,0 2,5 3,0

Time(min)
BGE: Sodium phosphate buffer 50 mM at pH 7.4 + 18 mM SDS; Uncoated fused silica
capillaries: 50 m i.d. and 48,5cm total length (8 cm effective length); Sandwich mode injection
CYP:S:CYP, each for 6 s; Voltage switch : +0,5 kV/-0,5 kV/+0,5 kV/-0,5 kV each for 7s;
Wainting time: 15 min; Migration voltage: - 25 kV; Detection: 320 nm.

Figure II.3.13. Influence of capillary temperature

EC
60

50
Absorbance (mAU)

40

30 NADPH

20 HC

C
10
B
A
0
0,0 0,5 1,0 1,5 2,0 2,5 3,0

Time (min)

BGE: Sodium phosphate buffer 50 mM at pH 7.4 + 18 mM SDS; Uncoated fused silica capillaries:
50 m i.d. and 48,5cm total length (8.5 cm effective length); Sandwich mode injection;
Temperature: 25 C; Voltage switch : +0,5 kV/-0,5 kV/+0,5 kV/-0,5 kV each for 7s; Wainting
time: 15 min; Migration voltage: - 25 kV; Detection: 320 nm.

Figure II.3.14. Influence of the plugs length on EC metabolization

32"
"
 II.3.3.2. Study of the enzymatic parameters [2]

When working with metabolic studies, the conversion rate of the substrate by the
enzymatic system must be optimized [3]. So it becomes crucial to perform the
experiments under conditions that ensure linearity which concerns incubation time and
enzyme concentration.

To begin with, the relation between incubation time and reaction velocity was
studied, maintaining the substrate and enzyme concentration constant (250 M and 200
pmol/mL). Four alternatives were compared, varying the incubation time at 10, 15, 30
and 60 minutes (Figure II.3.15.). On the other hand, the relation between the reaction
velocity and CYP1A1 concentration was determined by maintaining constant the
substrate concentration and the incubation time (250 M and 15 minutes). The enzyme
concentrations employed were 100, 150, 200 and 250 pmol/mL (Figure II.3.16.). The
best compromise between a short time of analysis and a good turnover for the substrate
was achieved by choosing an incubation time of 15 minutes and a CYP1A1
concentration of 200 pmol/mL for further studies.

14"

12"

10"
CAHC"

8"

6"

4"
0" 10" 20" 30" 40" 50" 60" 70"
!!!!!!!!!!!!!!!!!!Time!(min)!

BGE: Sodium phosphate buffer 50 mM at pH 7.4 + 18 mM SDS; Uncoated fused silica capillaries: 50 m i.d. and 48,5cm total
length (8.5 cm effective length); Sandwich mode injection; Temperature: 25 C; Voltage switch : +0,5 kV/-0,5 kV/+0,5 kV/-
0,5 kV each for 7s; Migration voltage: - 25 kV; Detection: 320 nm.

Figure II.3.15. Effect of the incubation time on HC formation

33"
"
 15"

13"

CAHC" 11"

9"

7"

5"
80" 100" 120" 140" 160" 180" 200" 220" 240" 260"

CYP1A1"concentration"(mg/mL)"

!!!!!!!!!!!!!!!!!
BGE: Sodium phosphate buffer 50 mM at pH 7.4 + 18 mM SDS; Uncoated fused silica capillaries: 50 m i.d. and 48,5cm
total length (8.5 cm effective length); Sandwich mode injection; Temperature: 25 C; Voltage switch : +0,5 kV/-0,5 kV/+0,5
kV/-0,5 kV each for 7s; WT: 15 min; Migration voltage: - 25 kV; Detection: 320 nm.

Figure II.3.16. Effect of the CYP1A1 concentration on the product formation

II.3.3.3. Design of experiment [2]

The final step in our development was the optimization of the electrophoretic
mixing, a key factor in EMMA procedures. Instead of using a mathematical method to
predict zone overlaps, the use of a design of experiment was chosen. This alternative is
due to a difference in conductivity between the reagent zones and BGE, the electric
field not being the same across all the capillary.

A few number of parameters influencing the metabolization rate were thus

investigated: the voltage mixing value (X1 ; three levels: 0.1, 0.55 and 1 kV) and the
voltage mixing time (X2; three levels: 2, 6 and 10 seconds). HC CA was proposed as a
response to maximize, Y. The response was modeled by the following equation:

Y= 0 + 1X1 + 2X2+ 12X1X2+ 11X11+ 22X22 + ,

Where 0 is the intercept, 1 and 2 are the main effect terms, 12 is the interaction
term, 11 and 22 are the quadratic terms and the error term.

34"
"
 For a better visualization of the results, a 3D plot was used (Figure II.3.17.).
The optimal mixing conditions were found to be 0.22 kV for the voltage mixing and, as
for the voltage mixing time, the best value was superior of the tested. The limit of 10
seconds was chosen with respect to the total time of analysis.

Figure II.3.17. Response surface of the effect of voltage mixing value and voltage
mixing time on HC CA and prediction profiles

The predicted condition was performed in triplicate and a value of 16.9 0.4
was obtained for the HC CA (Figure II.3.18.). The quality of this predictive model was
underlined by the experimental value, which falls within the confidence interval of 17.2
0.7. In addition, the RDSs for all the registred parameters ( HC CA, HC migration
time, EC CA, EC migration time) were lower than 2.1% , an excellent value considering
the complexity of our system (Table II.3.4.).

Table II.3.4. Repeatability in the optimal conditions

Area TM HC CAHC Area TMEC CAEC

HC EC
1st injection 10.25 0.59 17.28 50.99 0.82 62.11
2nd injection 10.04 0.60 16.88 50.87 0.82 62.11
3rd injection 10.13 0.61 16.58 49.20 0.80 61.27
Average 10.14 0.60 16.91 50.35 0.81 61.83
SD 0.10 0.01 0.35 1.00 0.01 0.48
RDS 1.00 1.65 2.07 1.98 1.21 0.78

35"
"
 Using a standard calibration curve, the 7-HC peak areas were converted into
concentrations, in order to determine the amount produced and the conversion rate.
Over the range of concentrations employed, 20-100 M, the regression equation was
found linear (y=0.2452x + 0.2837) , with a really high coefficient of determination ( r2
= 0.9999). With a measured concentration of HC (67.8 1.4 M), a volume of injected
plugs calculated according to the equation found in chapter II.2.4., an incubation time of
15 minutes and a CYP1A1 concentration of 200 pmol/mL, the reaction rate was found
to be 11.3 nmol of HC/min/nmoles of CYP1A1 in our optimal conditions.

To test our system efficiency, a comparison with the conventional offline

procedure was performed, maintaining all the experimental conditions identical (Figure
II.3.18.). A slightly higher HC CA was obtained after offline metabolization ( 20.0
0.1), compared with our inline value (16.9 0.4). The difference may result from the
fact that the mixing is performed electrophoretically and therefore may be not complete.
However, out method is fully automated, saving time and reagents.

II.3.4. Applicability [2]

II.3.4.1. Applicability to an in vitro model

The developed inline system may by useful for a more complex in vitro assay.
For this purpose, a test was performed using MC-induced liver microsomes (2mg/mL).
A triplicate was effectuated and the obtained results were comparable taking into
consideration the metabolite rate formation ( Figure II.3.18.). Once again, the selectivity
of our method was confirmed.

II.3.4.2. Applicability to CYP1A1 inhibitors screening

Knowing that CYP1A1 is a possible target for cancer treatment, our system
potency was tested regarding inhibition studies. A known inhibitor, apigenin, was
selected and a significant reduction of HC amount was noticed when our enzyme was
preincubated with 100 M apigenin, for 30 minutes at room temperature (Figure II.3.19.).

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 50 EC

40

Absorbance (mAU) 30
NADPH

20 HC

OFF-LINE
10
MC EMMA

CYP EMMA
0
0,0 0,5 1,0 1,5 2,0 2,5 3,0

Time (min)
BGE: Sodium phosphate buffer 50 mM at pH 7.4 + 18 mM SDS; Uncoated fused silica capillaries: 50 m i.d.
and 48,5cm total length (8.5 cm effective length); Temperature: 25 C; Voltage switch : +0,22 kV/-0,22
kV/+0,22 kV/-0,22 kV each for 10s; WT: 15 min; Migration voltage: - 25 kV; Detection: 320 nm.

Figure II.3.18. CYP1A1 vs. MC vs. Off-line metabolization

EC
40

NADPH
Absorbance (mAU)

30

Apigenin
related
20

HC With apigenin
10

Without apigenin

0
0,0 0,5 1,0 1,5 2,0 2,5 3,0

Time (min)
BGE: Sodium phosphate buffer 50 mM at pH 7.4 + 18 mM SDS; Uncoated fused silica capillaries: 50 m i.d.
and 48,5cm total length (8.5 cm effective length); Sandwich mode injection; Temperature: 25 C; Voltage
switch : +0,22 kV/-0,22 kV/+0,22 kV/-0,22 kV each for 10s; WT: 15 min; Migration voltage: - 25 kV;
Detection: 320 nm.

Figure II.3.19. Apigenin as CYP1A1 inhibitor

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 CONCLUSIONS

Since the efficacy and toxicity of drugs are closely related to their
pharmacokinetics, a good understanding of metabolic pathways is important at an early
stage of development. For a good quantification of all the processes involved, a potent
analytical methodology needs to be employed.
The present study describes the development of a fully automated incapillary
method to monitor CYP1A1 activity, a possible target for cancer therapy. After two
series of preliminary studies, the key factors were identified: incubation time, injection
mode, the concentration of the enzyme and substrate, voltage switch and voltage switch
time. Satisfying results were obtained using short-end injection procedure, employing
microsomes (2 mg/mL) in sandwich mode and applying rapid polarity switches for a
determined period of time (0.5 kV for 6 seconds).
The optimization stage gave rise to a baseline separation of the molecules of
interest (7-ethoxycoumarin, 7-hydroxycoumarin, and nicotinamide adenine dinucleotide
phosphate reduced) in less than 2 minutes. The optimization followed two pathways: the
improvement of incapillary metabolization procedure and the study of enzymatic
parameters.
Incapillary metabolization was significantly influenced by the length of the
injected plugs, the best results being obtained after injecting at -50 mbar for 6 seconds.
Moreover, a capillary temperature of 25 C and a final concentration of organic solvent
of 2.5% were settled, in order to maintain enzyme activity.
The turnover of the substrate was also optimized, by studying the impact of the
incubation time and the CYP1A1 concentration over the amount of produced
metabolite. An incubation time of 15 minutes and a CYP1A1 concetration of 200
pmol/mL were chosen for further studies.
In addition, the electrophoretic mixing proved to be another key issue of
integrated microanalysis. To the best of our knowledge, it was for the first time that the
mixing parameters were studied by a fully factorial design of experiment. The voltage
mixing value and the voltage mixing time were selected as parameters. The optimal
value predicted for the voltage mixing value was 0.22 kV, for a period of 10 seconds.
The experimental value (16.9 0.4) was found to fall within the confidence interval
(17.2 0.7), which underlines the quality of the predictive model. Taking into account
the measured concentration of hydroxycoumarin (67.8 1.4 M) , along with all the

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others values presented above, the reaction rate was found to be 11.3 nmoles of
hydroxycoumarin/min/nmoles of CYP1A1 in our optimal conditions.
Interestingly, the amount of 7-hydroxycoumarin obtained in the optimal
conditions (16.9 0.4) was found to be comparable to the one detected after
conventional offline metabolization (20.0 0.1 M). Finally, the potency of our system
to perform inhibition studies was demonstrated using 100 M apigenin as CYP1A1
inhibitor, a significant reduction of hydroxycoumarin amount being noticed.
It is noteworthy that the compatibility of our system with the use of surfactants
(MECK mode), ensures its applicability to a large range of molecules. In addition, the
miniaturization and the automatization of the process are the main advantages of
performing inline metabolization, since the enzymatic reaction, the separation, and the
detection are all performed in a single capillary. Besides, the reagents consumption is
drastically reduced due to the injection of few tens of nanoliters, with a very short time
of analysis.

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 REFERENCES

1. Hughes JP, Rees S, Kalindjian SB et al Principles of early drug discovery,

Br J Pharmacol, 2011, 162: 1239-1249.

2. Farca E, Servais AC, Lamalle C et al Fully automated electrophoretically

mediated microanalysis for CYP1A1 activity monitoring optimized by multivariate
approach, Electrophoresis, 2016, 37: 248-255.

3. Baranczewski P, Stanczak A, Sundberg K et al Introduction to in vitro

estimation of metabolic stability and drug interactions of new chemical entities in drug
discovery and development, Pharmacol Rep, 2006, 58: 453-472.

4. Nowak P, Woniakiewicz M, Kocielniak P Simulation of drug metabolism,

Trends in Analytical Chemistry, 2014, 59: 42-49.

5. Curcio R, Nicoli R, Rudaz S et al Evaluation of an in-capillary approach for

performing quantitative cytochrome P450 acitivity studies, Anal Bioanal Chem, 2010,
398: 2163-2171.

6. Go RE, Hwang KA, Choi KC Cythochrome P450 1 family and cancers, J

Steroid Biochem Mol Biol, 2015, 147: 24-30.

7. Walsh AA, Szklarz GD, Scott EE Human cytochrome P450 1A1 structure
and utility in understanding drug and xenobiotic metabolism, J. Biol. Chem, 2013, 288:
12932-12943.

8. Sankhwar M, Sankhwar SN Variations of CYP isoforms and bladder

cancer: a superfamily paradigm, Urol Oncol, 2014, 32: 33-40.

9. Androutsopoulos VP, Spyrou I, Ploumidis A et al Expression profile of

CYP1A1 and CYP1B1 enzymes in colon and bladder tumors, Plos One, 2013, 8 :
e82487.

10. Choudhury JH, Singh SA, Kundu S et al Influence of the CYP1A1 T3801C
Polymorphism on Tobacco and Alcohol-Associated Head and Neck Cancer
Susceptibility in Northeast India, Asian Pac J Cancer Prev, 2015, 16: 6953-6961.

40"
"
 11. Plant N Strategies for using in vitro screens in drug metabolism, Drug
Discov, 2004, 9: 328-336.

12. Jia L, Liu X The Conduct of Drug Metabolism Studies Considered Good
Practice (II) : In vitro experiments, Curr Drug Metab, 2007, 8: 822-829.

13. Donato MT, Castell JV Strategies and Molecular Probes to Investigate the
Role of Cytochrome P450 in Drug Metabolism, Clinical Pharmacokinetics, 2003, 42:
153-178.

14. Di L, Kerns EH, Hong Y et al Optimization of a higher throughput

microsomal stability screening assay for profiling drug discovery candidates, J Biomol
Screen, 2003, 8: 453-462.

15. Pearce RE, McIntyre CJ, Madan A et al Effects of freezing, thawing, and
storing human liver microsomes on cytochrome P450 activity, Arch Biochem Biophys,
1996, 331: 145-69.

16. Bjornsson TD, Callaghan JT, Einolf HJ et al The conduct of in vitro and
in vivo drug-drug interaction studies: a Pharmaceutical Research and Manufacturers of
America (PhRMA) perspective, Drug Metab Dispos, 2003, 31: 815-32.

17. Camilleri P Capillary electrophoresis. Theory and practice, CRC press,

Florida, 1993, 5-6.

18. European Pharmacopoeia 8th Edition, Chapter 2.2.47. Capillary

Electrophoresis, 2014, 83-88.

19. Wang X, Li K, Adams E et al Recent advances in CE-mediated

microanalysis for enzyme study, Electrophoresis, 2014, 35: 119-127.

20. Iqbal S, Rahman N, Iqbal J A capillary electrophoresis-based enzyme

assay for kinetics and inhibition studies of carbonic anhydrase, Anal Biochem, 2014,
444: 16-21.

21. Zhang J, Hoogmartens J, Schepdael AV Advances in CE-mediated

microanalysis: An uptade, Electrophoresis, 2008, 29: 56-65.

41"
"
 22. Zhang J, Hoogmartens J, Schepdael AV Advances in capillary electrophoretically
mediated microanalysis: An update, Electrophoresis, 2006, 27: 35-43.

23. Hai X, Yang B, Schepdael AV Recent developments and applications of

EMMA in enzymatic and derivatization reactions, Electrophoresis, 2012, 33: 211-227.

24. Bao J , Regnier FE Ultramicro enzyme assays in a capillary

electrophoretic system, J Chromatogr A, 1992, 608: 217-224.

25. Nowak P, Wozniakiewicz M, Wozniakiewicz P An overview of on-line

systems using drug metabolizing enzymes integrated into capillary electrophoresis,
Electrophoresis, 2013, 34: 2604-2614.

26. Van Dyck S, Van Schepdael A, Hoogmartens J Michaelis-Menten analysis

of bovine plasma amine oxidase by capillary electrophoresis using electrophoretically mediated
microanalysis in a partially filled capillary, Electrophoresis, 2001, 22: 1436-1442.

27. Okhonin V, Wong E, Krylov SN Mathematical model for mixing reactants

in a capillary microreactor by transverse diffusion of laminar flow profiles, Anal
Chem, 2008, 80: 7482-7486.

28. Krylova SM ,Okhonin V, Krylov SV Transverse diffusion of laminar flow

profiles-a generic method for mixing reactants in capillary microreactor, J Sep Sci,
2009, 32: 742-756.

29. Sanders BD, Slotcavage RL, Scheerbaum DL et al Increasing efficiency

of in-capillary electrophoretically mediated microanalysis reactions via rapid polarity
switching, Anal Chem, 2005, 77: 2332-2337.

30. Merola ET, Catherman AD, Yehl JB et al Determination of total

antioxidant capacity of commercial beverage samples by capillary electrophore`sis via
inline reaction with 2,6-dichlorophenolindophenol, J Agric Food Chem, 2009, 57:
6518-6523.

31. Stahl JW, Catherman AD, Sampath RK et al Investigating the effects of

conductivity on zone overlap with EMMA : Computer simulation and experiment,
Electrophoresis, 2011, 32: 1492-1499.

42"
"
 32. Pauwels J, Hoogmartens J, Schepdael AV Application of carbon nanotubes
for in-capillary incubations with cytochrome P450 enzymes, Electrophoresis, 2010, 31:
3867-3873.

33. Musheev MU, Filiptsev Y, Krylov SN Temperature difference between

the cooled and the noncooled parts of an electrolyte in capillary electrophoresis, Anal
Chem, 2010, 82: 86928695.

34. Glatz Z Application of short-end injection procedure in CE,

Electrophoresis, 2013, 34: 631-642.

35. Nemec T, Glatz Z Integration of short-end injection mode into

electrophoretically mediated microanalysis, J Chromatogr A, 2007, 1155: 206-213.

36. Terabe S Capillary Separation: Micellar Electrokinetic Chromatography,

Anal Chem, 2009, 2: 99-120.

37. Hancu G, Simon B, Rusu A et al Principles of Micellar Electrokinetic

Capillary Chromatography Applied in Pharmaceutical Analysis, Advanced
Pharmaeutical Bulletin, 2013, 3: 1-8.

38. Hai X, Nauwelaers T, Busson R et al A rapid and sensitive CE method

with field-enhanced sample injection and in-capillary derivatization for
selenomethionine metabolism catalysed by flavin-containing monooxygenases,
Electrophoresis, 2010, 31: 3352-3361.
39. Zhang J, Hoogmartens J, Schepdael AV "Kinetic study of cytochrome P450 by
capillary electrophoretically mediated microanalysis, Electrophoresis, 2008, 29: 3694-3700.

40. Asensi BL, Martn BY, Escuder GL et al " Fast evaluation of

enantioselective drug metabolism by electrophoretically mediated microanalysis:
application to fluoxetine metabolism by CYP2D6, Electrophoresis, 2013, 34: 3214-
3220.

41. Asensi BL, Martn BY, Escuder GL et al In-line capillary electrophoretic

evaluation of the enantioselective metabolism of verapamil by cytochrome P3A4, J
Chromatogr, 2013, 1298: 139-145.

43"
"
 42. Ying KH, Thormann W Enantioselective capillary electrophoresis for the
assessment of CYP3A4-mediated ketamine demethylation and inhibition in vitro,
Electrophoresis, 2012, 33: 32993305.

43. Mason PE, Schildt DC, Strein TG In-capillary determination of creatinine

with electrophoretically mediated microanalysis: characterization of the effects of
reagent zone and buffer conditions, J Chromatogr A, 2009, 1216: 154-158.

44. Shimada T, Tsumura F, Yamazaki H Prediction of human liver

microsomal oxidations of 7-ethoxycoumarin and chlorzoxazone with kinetic parameters
of recombinant cytochrome P-450 enzymes, Drug Metab Dispos, 1999, 27: 12741280.

45. Breadmore MC Electrokinetic and hydrodynamic injection: making the

right choice for capillary electrophoresis, Bioanalysis, 2009, 1: 889-894.

46. Ball DW, Hill JW, Scott RJ " The Basics of General, Organic, and Biological
Chemistry, v. 1.0,"Chemistry Department Books, New York, 2011, 1056-1059.

44"
"