Expression of drug transporters in intestine and blood

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.