Date of Award

12-2007

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Research Advisor

Bernd Meibohm, Ph.D.

Committee

Michael Christensen, Pharm.D. Stephanie Phelps, Pharm.D. Erin G. Schuetz, Ph.D. Charles Ryan Yates, Pharm.D., Ph.D.

Keywords

ontogeny, liver drug transporter, nuclear receptor, multidrug resistance protein, ceftriaxone

Abstract

Members of the ATP-binding cassette (ABC) family of drug transporter proteins translocate various endogenous and exogenous substrates across intra- and extracellular membranes. Two specific ABC transporters, the multidrug resistance 1/P-glycoprotein (MDR1/P-gp) and the multidrug resistance protein 2 (MRP2), serve as major hepatic transporters that mediate the biliary excretion of various organic anions and cations along with glutathione-, glucuronate-, or sulfate-conjugates of several drug substrates. However, very little is known about the expression of these transporters in the early infant and childhood ages of human development. We, therefore, characterized the ontogeny of these transporters by measuring their gene and protein expression. Furthermore, we also characterized the ontogeny of four nuclear receptors through measurement of their gene expression. Nuclear receptors belong to a highly conserved gene superfamily of transcriptional factors that regulate the expression of their target genes, many of which include members of the ABC transporters. We identified four main nuclear receptors that have been associated with the modulation of gene expression of MDR1/P-gp and MRP2 to include pregnane X receptor (PXR), constitutive androstane receptor (CAR), farnesoid X receptor (FXR), and hepatic nuclear factor 4a (HNF4alpha). Our study revealed lower gene expression of MDR1/P-gp and MRP2 in the early infant period of development. We also identified a lower protein expression of MRP2 in the early infant period. For the nuclear receptors, we found significantly lower expression levels for PXR and FXR in the early periods of development. Positive correlations were established between the nuclear receptors PXR and FXR to the ABC transporters MDR1/P-gp and MRP2. HNF4a, which has been described as a master regulator of other nuclear receptors, was determined to be positively correlated with PXR and CAR.

Due to the various challenges associated with conducting scientific research in newborns, infants and children and obtaining tissue samples in this population, we wished to establish the ontogeny of the human equivalent of the ABC transporters and nuclear receptors in a rat model. As such, we determined the ontogeny of Mdr1a/1b, Mrp2, Pxr, Car, Fxr, and Hnf4alpha from rat liver samples in seven different postnatal ages in order to compliment our human results with more selected spacing of age and sample numbers in our rat samples. Similar to our human pediatric liver findings, rat liver Mdr1a/1b and Mrp2 gene expression appeared limited in the first week of life and increased thereafter. The same findings were true for the rat P-gp and Mrp2 protein samples. The rat nuclear receptors Pxr, Car, and Fxr all exhibited a positive correlation with both Mdr1a/1b and Mrp2 gene expression. Furthermore, Hnf4a was also positively correlated with all three nuclear receptors.

In a subsequent experiment, we aimed to explore the functional consequences in changes in drug transporter expression on the pharmacokinetics of their drug substrates. Ceftriaxone, a third generation cephalosporin antibiotic frequently used in pediatric pharmacotherapy, was selected as our model compound. The pharmacokinetics of a single intravenous dose of ceftriaxone was compared between wild-type (WT) Wistar rats as compared to a mutant strain of Mrp2-deficient (TR-) rats. We chose the use of TR- rats as a surrogate animal to represent the immaturity of Mrp2 early on in development as was evident from our rat and human liver samples model. Using the TR- rat model, we reported significantly longer elimination half-life (t1/2) (p < 0.05), undetectable amounts of drug in the feces (p < 0.05), and an increase in the urinary excretion of unchanged drug (p < 0.001) in the TR- as compared to the WT rats.

In summary, these results demonstrate that the hepatic ABC transporters MDR1/P-gp and MRP2 are differentially expressed during childhood maturation and suggest that the observed reduced expression of MRP2 in human liver in early infancy may result in clinically significant differences in the disposition of medications used in pediatric pharmacotherapy.

DOI

10.21007/etd.cghs.2007.0312

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