Expression of drug transporters in intestine and blood

Abstract

Proteins that are capable to transport molecules across membranes are fundamental for the accurate functioning of the body. Many diseases have their cause in a dysfunction of a particular transport protein. Membrane transporters are involved in the absorption, distribution, metabolism, and excretion of endogenous and ingested substances, but for numerous transport proteins their substrates and physiological roles are still unknown or hypothetical. Most transporters exhibit a high specificity for their natural substrates. However, some transporters show broad substrate specificity, thereby translocating a large variety of substances including drugs. Consequently, the expression of so-called drug transporters can influence the pharmacokinetics of administered drugs by controlling their oral absorption, their distribution within the body, and their elimination through excretory organs. Furthermore, over-expression of particular drug transporters can lead to a decreased drug bioavailability. The reduced drug concentrations in blood and in tissues can even result in a phenotype of drug resistance. This phenomenon is often observed in patients with cancers. However, therapy resistance is also a well-known problem in other diseases such as inflammatory bowel disease (IBD). Approximately 50% of patients with Crohn`s disease and 20% of patients with ulcerative colitis require other therapeutic strategies due to inefficient steroid treatment. Many of these patients need surgery as a result of therapy resistance. But the underlying mechanisms of therapy resistance in IBD patients are poorly understood. The aim of this thesis was to assess the general expression of transporters in humans. The main focus was the intestine as an important site of drug absorption. Furthermore, in vitro experiments using intestinal cell lines were performed to evaluate alterations in transporter expression by drugs and endogenous compounds. This knowledge can help to assess the impact of these transporters on 1) the oral bioavailability of drugs, 2) therapy resistance, 3) possible drug-drug interactions. Initially, a method was developed to accurately quantify the expression of transporters using realtime PCR (TaqMan® analysis, chapter 2). Thus, a standard for each gene of interest was synthesized and quantified in order to compose standard curves with known amounts of PCR templates. Consequently, for each transporter the gene-expression could be expressed as absolute mRNA transcript number. This method was used in all projects where mRNA expressions were analyzed. The general expression of drug transporter mRNA along the human intestinal tract was studied in biopsies from 10-14 healthy volunteers (chapter 3). Biopsies were taken from the duodenum, the terminal ileum, and from the proximal to the distal colon (ascending, transverse, descending, and sigmoid colon). Site-specific mRNA expressions for MDR1 and MRP1-5 (chapter 3.1), BCRP (chapter 3.2), and ASBT (chapter 3.3) were shown. These data can be useful in developing new targeting strategies for enteral drug delivery. Additionally, the transporter expression obtained in these healthy control patients can be compared with the transporter expression in IBD patients in further studies. This might help to elucidate the role of transporters in IBD. Using in vitro experiments, we investigated whether budesonide, an often-used glucocorticoid in patients with IBD, might affect the expression of drug transporters (chapter 4.1). A selective induction of MDR1 on mRNA and protein level was detected in a human intestinal cell line. Since budesonide is also a P-gp substrate, this induction might be one reason for the steroid resistance that is often observed in IBD patients treated with glucocorticoids. Thalidomide is an “old” drug that is increasingly used as an adjuvant therapy in malignant and inflammatory diseases, including IBD. Therefore, this drug was screened for possible interactions with P-gp (chapter 4.2) and MRP2 (chapter 4.3) by performing induction-, inhibition-, and transport-assays. Thalidomide showed no potential for interactions regarding these two drugefflux transporters. Furthermore, a HPLC method for the determination of thalidomide enantiomers in blood was developed (chapter 5). This sensitive method can be applied in prospective clinical trials where the efficacy of thalidomide is further investigated. In a study, including vasospastic persons with increased Endothelin-1 plasma levels, the expression of MDR1 and MRP1-5 in isolated blood mononuclear cells was determined (chapter 6). Vasospastic persons differed from healthy controls in their expression pattern of transporter proteins. They showed a significant decrease in their expression of MDR1, MRP2, and MRP5 mRNA when compared to controls. This might be an indirect effect of elevated ET-1 levels and this could explain the enhanced drug-sensitivity reported by these patients. In a further project, the release of mitomycin C from collagen implants was determined using a newly developed HPLC method (chapter 7). In this study it was clearly shown that commercially available collagen implants could be loaded with MMC, and could subsequently release it. The pharmacokinetics of this relationship is determined in vitro.