Date of Award

5-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Track

Pharmaceutics

Research Advisor

Bernd Meibohm, Ph.D.

Committee

Sarka Beranova-Giorgianni, Ph.D. Michael L. Christensen, Pharm.D. Alex Sparreboom, Ph.D. C. Ryan Yates, Pharm.D., Ph.D.

Keywords

Drug Transport, Fexofenadine, OATP1B1, OATP1B3, Ontogeny, Pediatrics

Abstract

As part of the drug disposition process (absorption, distribution, metabolism, excretion), an often overlooked aspect is transport. In order for drugs to be metabolized and excreted from the body they go through the liver or other drug removal organs. For drugs that are polar or are large they must rely upon transport mechanisms to transport them across the biomembranes of the drug removal organs. OATP1B1 and OATP1B3 are transporters on the sinusoidal membrane of the liver which work in concert with the drug metabolizing enzymes as part of the drug removal process. It is known that the development of each drug metabolizing enzyme follows a unique pattern that is determined in part by age. This is not unique to drug metabolizing enzymes or even drug disposition processes. All aspects of physiologic development are governed in part by age from gastric pH, and kidney function, to skin thickness. With the knowledge that age is a major determinant of expression of drug metabolizing enzymes it was hypothesized that age would also impact the expression of drug transporters. The developmental protein expression pattern of the closely related hepatic uptake transporters, OATP1B1 and OATP1B3, was examined in human liver tissue samples. Samples were obtained from donors aged 14 days to 12 years. Western blot analysis revealed a very intriguing pattern. OATP1B3 showed very high expression at birth but tapered down to minimal expression by 6 months of age. The intriguing aspect of this data was that the protein expression level of OATP1B3 started to increase during the pre-adolescent period. When comparing the protein expression levels in infants less than 3 months to children aged 6-12 years, protein expression was 237% higher in the infants. The expression decreased to 33% in the 3 months to 2 years group as compared to the 6-12 years group with the 2-6 years group starting to increase expression to 50%. OATP1B1 showed large inter-individual variability but failed to show a significant difference in protein expression amongst the age groups examined. While ontogeny is a very important part of overall development, genetics is also very important. The term developmental pharmacogenetics describes the inter-play between age and genetics, as phenotypes can only be expressed if physiological development is at a point where it is possible to be seen. In order to learn more about the expression of OATP1B1 and OATP1B3 several common SNPs were examined, OATP1B1 388 A→G and 521 T→C as well as OATP1B3 334T→G and 699G→A. All four SNPs were found to be in Hardy-Weinberg equilibrium. The allele frequencies for each SNP were in agreement with previously published reports. OATP1B3 334T→G and 699G→A were in complete linkage disequilibrium. The linkage disequilibrium that was found between the SNPs in OATP1B3 and OATP1B1 388 A→G was a novel finding that deserves further exploration. Examination of protein expression and each SNP failed to show any association between the two which also deserves further exploration with a larger number of samples to confirm. Lastly, the functional consequences of the loss of OATP1B activity was explored using the Oatp1b2-/- mouse model. Oatp1b2-/- is the rodent member of the OATP1B family and shares greater than 65% homology to the human members. Using fexofenadine as a probe for Oatp1b2 activity, it was found that Oatp1b2-/- mice had 2.1 fold higher exposure as compared to the wild-type mice. The Oatp1b2-/- also had 55% of the fexofenadine clearance and 63% the volume of distribution as compared to wild-type mice. These findings underscore the importance that Oatp1b activity has in determining exposure, and thus efficacy and potential toxicity, to substrates. Drug transport is a very important part of drug disposition that has yet to be extensively studied especially in terms of drug uptake. The following studies examined the developmental expression patterns of hepatic uptake transporters OATP1B1 and OATP1B3, common genetic variations of the transporters, as well as the consequences the loss of OATP1B activity has on substrates. Understanding the developmental expression pattern of OATP1B will allow for better dosing strategies for patients, particularly infants and children who rarely participate in clinical trials. Better dosing strategies ultimately lead to improved outcomes and reduced toxicity in the pediatric population.

ORCID

http://orcid.org/0000-0002-2147-6943

DOI

10.21007/etd.cghs.2017.0440

Comments

One year embargo expires May 2018.

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