1. Effects of Anti-Epileptic Drugs (AED), Phenytoin and Carbamazepine, on Calcium Transport in Caco-2 Cells, an Immortalized Human Intestinal Cell Line Melinda von Borstel Senior in Nutrition Science/ Pre- Pharmacy

2. The Impact of Epilepsy l l The Statistics: - > 2 million people in the U.S. and about 50 million people worldwide - Costs reach an estimated $12.5 billion/ year

3. Benefits of AED Goals of Therapy o o Stop seizures o o No unwanted side effects

4. Side Effects of AED l l Fatigue, abdominal discomfort, dizziness, or blurred vision during the first weeks l l Rash, inflammation or failure of the liver or pancreas, a serious reduction in the number of white blood cells (needed to fight infection), a serious reduction in the number of platelets (needed to control bleeding) l l Osteomalacia with certain AED

5. Anti-epileptic Drugs and Osteomalacia l l Adverse effects on bone density reported since the early 1960’s l l Phenytoin and carbamazepine most frequently associated with osteomalacia l l Newer agents (e.g. topiramate, lamotrigine, gabapentin) appear to be less causitive of osteomalacia

6. Proposed Mechanisms for Bone Loss With AED Treatment

7. Hypothesis l l My hypothesis is that phenytoin and carbamazepine will inhibit calcium transport from the apical to the basolateral side of Caco-2 cells grown on semi-permeable supports.

8. Studying Impaired Calcium Absorption Intestinal lumen Blood flow Nutrients Tight junctions Apical side Basolateral side Epithelial Cells

9. Why Use Caco-2 Cells as an In Vitro Model? l l Previously used to study oral bioavailability l l Caco-2 Cells are commonly used to study drug transport and intestinal calcium transport

10. Studying Impaired Calcium Absorption Using Caco-2 Cells

11. Basolateral Side Apical Side The Caco-2 Model Caco-2 Cells

12. Methods to Quantify Caco-2 Permeability with Drug Treatment l l Caco-2 cells are grown to confluency and maintained in culture until tight junctions form between the cells as confirmed by a transepithelial electrical resistance of >200 ohms. l l Calcium transport from the apical to basolateral side of the polar monolayer is monitored by spiking the apical cell culture medium with radioactive Ca45 at time zero. l l Calcium transport is quantified by sampling the basolateral medium and measuring by liquid scintillation spectrometry the amount of Ca45 radioactivity present at varying times up to 2.66 hours post treatment (established empirically as the time frame for linear transport conditions). l l Calcium permeability as a function of calcium transport over time is calculated under conditions of calcium homeostasis (equal starting calcium concentrations in apical and basolateral medium at time zero). The effects of various concentrations of phenytoin and carbamazepine on calcium permeability will be quantified. A dose response relationship between drug concentration and calcium permeability will be established. Ca++ Drug Basolateral Side pH 7.4 Apical Side pH 6.8 45Ca**

13. % Calcium Transported over Time with Phenytoin

14. Caco-2 Permeability with Phenytoin Treatment

15. % Calcium Transported over Time with Carbamazepine

16. Caco-2 Permeability with Carbamazepine Treatment

17. Preliminary Data l l Preliminary data have revealed an effect of both phenytoin and carbamazepine on calcium transport in Caco-2 cells compared to vehicle- treated controls

18. Further Investigation *Establish the time course necessary to affect Calcium permeability *Tests to find cause of variability

19. Special Thanks To:  Dr. Theresa Filtz  Dr. Rosita Rodriguez  John Mata  Howard Hughes Medical Institute