Lamotrigine Metabolism in the Human Placenta

Lindsay Samuel

Mentor: Phillip M. Gerk, PharmD, PhD Department of Pharmaceutics

Joseph K. Ritter, PhDDepartment of Pharmacology/Toxicology

Introduction

Many pregnant women have to take medication during pregnancy.

For many different reasons Example: Women

with seizure disorders

http://www.dsf.health.state.pa.us/health/cwp/view.asp?a=179&Q=237058&

Clinical Problem and Drug Development Medications may have adverse effects on

the fetus. There are safety concerns of including

pregnant women in clinical drug trials. Pharmaceutical companies study animal

pharmaceutical toxicology and test drugs in pregnant rats and mice,

The data from these studies cannot be directly transferred over to human patients Human placenta anatomically differs from

rats and mice

In the lab…

In Dr. Gerk’s and Dr. Ritter’s lab, we are studying Lamotrigine metabolism.

Lamotrigine is an anti-seizure medication, which is somewhat safe in pregnancies

The objective in the lab is to determine how the placenta handles Lamotrigine.

Placenta

“Before We Are Born: Basic Embryology and Birth Defects”, Moore, 1974.

Enzymes

UDP-glucuronosyltransferase (UGT) family of enzymes found in the liver and other tissuesUGT2B7 is also expressed in the placenta

Glucuronidation of drugsExamples:

• Morphine: In a baboon model, the placenta makes major contributions to morphine clearance.

• Lamotrigine LTGLTG-Gluc

Lamotrigine

Indications Bipolar disorder Epilepsy

Mechanism Unknown, but may have an effect on sodium

channels Pharmacokinetics

Absorption: 98% bioavailability Distribution: mean apparent volume of distribution

following oral administration 0.9-1.3 L/kg Metabolism: glucuronidation is the major elimination

pathway (86% of dose) Excretion: 94% urine, 2% feces

Metabolism

UGT1A4 and UGT2B7 are the major isoforms involved

UGT2B7 is expressed in the placenta Clearance of lamotrigine is doubled in

pregnant women Clearance is also increased when

used with hormonal contraceptives UGT2B7 is inhibited by valproic acid

Research Questions

1. What role does placental UGT play in detoxifying drugs (like LTG)?

2. To what extent do anti-seizure drugs (like LTG) inhibit UGT activity?

My Research Proposal

Use 4-MU to establish UGT2B7 glucuronidation activity4-MU 4-MUG using UGT-2B7

Probe used to set up a procedure to detect LTG-Gluc formation in placental samples

Methods: Setup

4-MU 4-MUG Microplate detection method to

quantitatively determine the disappearance of 4-MU through diminishing fluorescence over time

4-MUG does not fluoresce with as much intensity and at a different wavelengths as 4-MU

Methods: Procedure

Variables: +/- protein (expressed UGT 2B7, rat liver microsomes,

human placental microsomes) +/- uridine diphosphoglucuronic acid (UDPGA), magnesium chloride, alamethicin 4-methylumbelliferone (4-MU) Buffer Time temperature

Taken from: http://www.bdbiosciences.com/discovery_labware/products/display_product.php?keyID=515

Results

Emission Spectra

300 350 400 450 500 550 6000

50

100

150

200Blank4-MUG4-MU

wavelength (nm)

Inte

nsi

ty

Excitation Spectra

275 300 325 350 375 400 425 450 4750

50

100

150

2004-MU4-MUG

Wavelength (nm)

Inte

nsity

Results

Human placenta Microsomes

0 10 20 30 40 50 60 700

250

500

750

1000

1250

With UDPGAWithout UDPGA

*

Time

Flu

ore

scen

ce

Methods: Setup

LTG LTG-Gluc Detection by reversed phase HPLC UV monitoring at 254 nm

Methods: Procedure

Variables: +/- protein (expressed UGT 2B7, rat liver

microsomes, human placental microsomes) +/- uridine diphosphoglucuronic acid

(UDPGA), magnesium chloride alamethicin Lamotrigine Buffer Time Temperature

Results

Standard Curve 070629

0 25 50 75 100 1250

50000

100000

150000

200000

250000

300000

350000

r2=0.9995

[Lamotrigine] (M)

Pea

k A

rea

(254

nm

)

Standard Curve 070629

0 25 50 75 100 1250

10000

20000

30000

40000

r2=0.9988

[Lamotrigine] (M)P

eak

Hei

gh

t (2

54n

m)

Results254 Peak Height

0

1000

2000

3000

4000

Protein

Pe

ak H

eig

ht

(25

4n

m)

at

tim

e=

2.2

-2.4

min

ute

s

Results254 Peak Height

0

1000

2000

3000

4000

Peak H

eig

ht

(254n

m)

at

tim

e=

2.2

-2.4

min

ute

s

Future Study

Improve HPLC method- to remove interfering peaks (background)

14C-UDPGA LTG-Gluc*Confirm HPLC resultsABC transporter substrate?

Goals

To hopefully understand how the human placenta handles drugs

To clinically determine which medications will be safe for women without dangerous clinical trials

References

Tatum, Expert Rev Neurother 2006; 6: 1077-86. Rowland et al, Drug Metab Disp 2006; 34: 1055-62 "Lamotrigine." Facts & Comparisons 4.0. 2007. Wolters Kulwer

Health INC.. 19 Jul 2007 <http://online.factsandcomparisons.com/monodisp.aspx?book=dfc&monoid=fandc-hcp11195&searched=lamotrigine>.

Christensen et al. Epilesia 2007; 48: 484-9

Acknowledgements

Dr. Phillip Gerk Dr. Joseph Ritter Fay Kessler

QUESTIONS?