Role of Cation- Selective Apical Transporters in Paracellular Absorption
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A NOVEL MECHANISM FOR INTESTINAL ABSORPTION OF THE TYPE II DIABETES DRUG METFORMIN: ROLE OF CATION- SELECTIVE APICAL TRANSPORTERS IN PARACELLULAR ABSORPTION William Ross Proctor III A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the School of Pharmacy. Chapel Hill 2010 Approved by Advisor: Dhiren R. Thakker, Ph.D. Chairperson: Moo J. Cho, Ph.D. Reader: James M. Anderson, M.D., Ph.D. Reader: Kim L.R. Brouwer, Pharm.D., Ph.D. Reader: Joseph W. Polli, Ph.D. Reader: Zhiyang Zhao, Ph.D. © 2010 William Ross Proctor III ALL RIGHTS RESERVED ii ABSTRACT William Ross Proctor III: A Novel Mechanism for Intestinal Absorption of the Type II Diabetes Drug Metformin: Role of Cation-selective Apical Transporters in Paracellular Absorption (Under the direction of Dhiren R. Thakker, Ph.D.) Metformin, a widely prescribed anti-hyperglycemic agent, is very hydrophilic with net positive charge at physiological pH, and thus should be poorly absorbed. Instead, the drug is well absorbed (oral bioavailability of 40-60%), although the absorption is dose- dependent and variable; the drug accumulates in enterocytes during oral absorption. To date, the transport processes associated with the intestinal absorption of metformin are poorly understood. This dissertation work describes an unusual and novel intestinal absorption mechanism for metformin. The absorption mechanism involves two-way transport of metformin across the apical membrane of enterocytes that is mediated by cation-selective transporters, and facilitated diffusion across the paracellular route, working in concert to yield high and sustained absorption Metformin absorption was evaluated in the well established model for intestinal epithelium, Caco-2 cell monolayers. Metformin was efficiently transported across the apical membrane by bidirectional cation-selective transporters; however, the drug accumulated in the cells due to inefficient egress across the basolateral membrane. Consequently, the absorptive transport was almost exclusively through the paracellular route; however, the paracellular transport contained a distinct saturable component. iii Evidence is presented to show that the mechanism responsible for the observed saturable paracellular transport involves electrostatic interactions between positively charged metformin and negatively charged amino acid residues on the pore-forming tight-junction protein, claudin-2. Treating Caco-2 cells with the active metabolite of vitamin D3, 1,25- dihydroxyvitamin D3, selectively induced claudin-2 in preference to other tight junction proteins, and concurrently increased paracellular transport of metformin. Overexpression of claudin-2 in renal epithelial cells, LLC-PK1, caused size-dependent increase in paracellular transport of small organic cations, further supporting the role of claudin-2 in facilitating paracellular transport of hydrophilic cationic compounds. By employing a novel chemical inhibition scheme, it was revealed that both the organic cation transporter 1 (hOCT1) and the plasma membrane monoamine transporter (PMAT) were involved in the apical uptake/efflux of metformin in Caco-2 cell monolayers. Taken together, these results suggest a novel mechanism to explain how a hydrophilic cation like metformin is absorbed efficiently, though it uses the inefficient paracellular route for absorption. It is hypothesized that metformin is taken up into enterocytes via apical cation-selective transporters, hOCT1 and PMAT, and accumulates in the cells because of inefficient basolateral egress due to the lack of cation-selective efflux transporters. At each segment of the intestine, a small fraction of the metformin dose is absorbed via the paracellular route, facilitated by claudin-2, while a significant portion of the dose is taken up into the cells. Drug is then effluxed back into the lumen as the dose of the drug travels forward, taken up into distal enterocytes, or absorbed through the paracellular space. The apical transporters function to sequester the drug and allow for multiple opportunities to be absorbed by the paracellular route; thus, increasing the residence time in the intestine iv enabling efficient absorption. This dissertation work provides novel insights into the mechanisms associated with intestinal absorption and accumulation of metformin. The absorption mechanism proposed can account for the sustained high exposure of metformin achieved in the primary pharmacological organ, the liver, via the portal circulation. Additionally, the mechanisms proposed here can account for the possible role of the intestine in the pharmacology, gastrointestinal side effects, and adverse events of metformin. v ACKNOWLEDGEMENTS I am incredibly grateful for my advisor Dr. Dhiren R. Thakker for his continued support, mentorship, patience, and friendship throughout my graduate training. I am deeply indebted to all my committee members, Drs. James M. Anderson, Kim L.R. Brouwer, Moo J. Cho, Joseph W. Polli, and Zhiyang Zhao for their time, suggestions, and valuable insight. I would especially like to thank James M. Anderson for his expertise and generous resources regarding the claudin studies presented in this dissertation. I would also like to acknowledge all of the faculty and students at the Eshelman School of Pharmacy for making my experience here memorable and rewarding. In particular I would like to thank Drs. Arlene Bridges, Mary F. Paine, and Craig Lee for opening up their laboratories and resources to me during my graduate training. This dissertation work was possible by generous support from Amgen, Inc. and the PhRMA Foundation in the form of pre-doctoral fellowships. I would like to express my deepest gratitude to my family and friends for helping me through this long journey. In particular I would like to thank my mother Alice C. Proctor for her unwavering love, support, and encouragement. Additionally, I would like to thank my brother and best friend, David Proctor, for always being there for me. I would like to acknowledge my sister Emily Kenning for keeping me grounded and for continually taking care of our family. I also would like to acknowledge my father Ross Proctor for encouraging me to explore and experience the world. Lastly, I would like to thank my future wife, Julie M. Bessette, for her love, support, and understanding throughout this difficult time. I am extremely fortunate to have her in my life. vi TABLE OF CONTENTS Page LIST OF TABLES................................................................................................................... xi LIST OF FIGURES ................................................................................................................ xii LIST OF ABBREVIATIONS................................................................................................ xiv Chapter 1. INTRODUCTION ...............................................................................................................1 A. INTRODUCTION .........................................................................................................2 B. PATHOLOGY AND PATHOGENESIS OF TYPE II DIABETES..............................3 C. METFORMIN PHARMACOLOGY.............................................................................4 1. Clinical Effects.........................................................................................................4 2. Insulin Sensitivity and Insulin Resistance ...............................................................6 3. Inhibition of Hepatic Gluconeogenesis....................................................................7 4. Intestinal Glucose Utilization ..................................................................................7 5. Peripheral Glucose Uptake in Skeletal Muscle........................................................8 6. Adipose Tissue and Insulin Resistance....................................................................9 7. Off-Label Indications and Emerging Therapeutic Areas.......................................10 D. ACTIVATION OF AMP-ACTIVATED PROTEIN KINASE (AMPK) ....................11 1. Overview of AMPK...............................................................................................11 2. AMPK Activation and Downstream Events..........................................................13 vii 3. Inhibition of Complex I of the Mitochondrial Respiratory Chain .........................16 E. METFORMIN PHARMACOKINETICS....................................................................18 1. Clinical Pharmacokinetics .....................................................................................18 a. Intravenous Administration .............................................................................19 b. Peroral Administration.....................................................................................20 2. Intestinal Disposition and Absorption ...................................................................21 3. Distribution ............................................................................................................22 4. Elimination.............................................................................................................24 a. Renal Clearance...............................................................................................24 b. Non-Renal Clearance.......................................................................................24