Transcript Summer Research: - Virginia Commonwealth University

Lamotrigine Metabolism
in the Human Placenta
Lindsay Samuel
Mentor: Phillip M. Gerk, PharmD, PhD Department of Pharmaceutics
Joseph K. Ritter, PhD
Department 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 tissues
 UGT2B7 is also expressed in the placenta
Glucuronidation of drugs
 Examples:
• Morphine: In a baboon model, the placenta
makes major contributions to morphine
clearance.
• Lamotrigine LTGLTG-Gluc
Lamotrigine
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

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.
2.
What role does placental UGT play
in detoxifying drugs (like LTG)?
To what extent do anti-seizure drugs
(like LTG) inhibit UGT activity?
My Research Proposal

Use 4-MU to establish UGT2B7
glucuronidation activity


4-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
Excitation Spectra
200
200
4-MU
4-MUG
100
Blank
4-MUG
4-MU
150
Intensity
150
Intensity
Emission Spectra
100
50
50
0
275 300 325 350 375 400 425 450 475
Wavelength (nm)
0
300
350
400
450
500
wavelength (nm)
550
600
Results
Human placenta Microsomes
Fluorescence
1250
1000
*
750
Without UDPGA
With UDPGA
500
250
0
0
10
20
30
40
Time
50
60
70
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
Standard Curve 070629
40000
Peak Height (254nm)
Peak Area (254nm)
350000
300000
250000
200000
r2=0.9995
150000
100000
50000
0
0
25
50
75
[Lamotrigine] (M)
100
125
30000
r2=0.9988
20000
10000
0
0
25
50
75
[Lamotrigine] (M)
100
125
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t
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D
ith
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tU A
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um
H
Peak Height (254nm)
at time= 2.2-2.4 minutes
Results
254 Peak Height
4000
3000
2000
1000
0
Protein
H
PM
H
PM
D
PG
U
A
ou
;B
tU
uf
D
PG
fe
rw
A
ith
ou
tU
D
PG
B
uf
A
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rw
ith
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D
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ith
rw
rw
ith
;B
uf
fe
uf
fe
;B
;L
TG
;L
TG
PM
H
Peak Height (254nm)
at time= 2.2-2.4 minutes
Results
254 Peak Height
4000
3000
2000
1000
0
Future Study
Improve HPLC method- to remove
interfering peaks (background)
 14C-UDPGA  LTG-Gluc*

Confirm HPLC results
 ABC 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
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References
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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?