1. 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

2. 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&

3. 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

4. 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.

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

6. 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

7. 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

8. 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

9. 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?

10. 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

11. 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

12. 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

13. Results

15. Methods: Setup LTG  LTG-Gluc Detection by reversed phase HPLC UV monitoring at 254 nm

16. Methods: Procedure Variables: +/- protein (expressed UGT 2B7, rat liver microsomes, human placental microsomes) +/- uridine diphosphoglucuronic acid (UDPGA), magnesium chloride alamethicin Lamotrigine Buffer Time Temperature

17. Results

20. Future Study Improve HPLC method- to remove interfering peaks (background) 14 C-UDPGA  LTG-Gluc* Confirm HPLC results ABC transporter substrate?

21. Goals To hopefully understand how the human placenta handles drugs To clinically determine which medications will be safe for women without dangerous clinical trials

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

23. Acknowledgements Dr. Phillip Gerk Dr. Joseph Ritter Fay Kessler

24. QUESTIONS?