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

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

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

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

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

Proposed Mechanisms for Bone Loss With AED Treatment

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.

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

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

Studying Impaired Calcium Absorption Using Caco-2 Cells

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

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 Ca**

% Calcium Transported over Time with Phenytoin

Caco-2 Permeability with Phenytoin Treatment

% Calcium Transported over Time with Carbamazepine

Caco-2 Permeability with Carbamazepine Treatment

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

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

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