ABSTRACT

TARGETING DRUG TRANSPORTERS AS A NOVEL APPROACH FOR PLATINUM-BASED ANTICANCER THERAPY

Shuanglian Li

Tissue specific targeting by design and development of influx transporter targeted compounds represents one effective approach for increasing efficacy and reducing toxicity of drugs. The overall goal of this dissertation research was to test the hypothesis that organic cation transporter 1 (OCT1) can be used as a target to deliver platinum-based chemotherapeutics to tumors with the goal of enhancing the efficacy and reducing the toxicity of these agents.

As a first step in evaluating the potential of OCT1 as a valid drug delivery target we determined the effect of OCT1 on the disposition of pyroplatin (a potent OCT1 substrate) under normal physiological OCT1 expression level in vitro and in vivo. Studies in primary hepatocytes and Oct1 knockout mice demonstrated that Oct1 plays a role in mediating the transport of pyroplatin into the Oct1 expressing tissues and accordingly its pharmacokinetics.

The goal of the second part of this dissertation was to determine the role of OCT1 in response to pyroplatin. In particular the role of OCT1 in the toxicity and antitumor effect of pyroplatin was intensively investigated using genetically modified mice. The data supported the concept that by targeting drug influx transporters, off-target toxicities can be greatly spared. Although OCT1 mediated the accumulation of pyroplatin in hepatocellular carcinoma, a benefit on overall survival of the mice was not observed. Further investigation is needed to elucidate the mechanisms of resistance of hepatocellular carcinoma to pyroplatin and other platinum based chemotherapies.

In the final part of this dissertation, the role of OCT1 in the disposition and drug action of oxaliplatin (a moderate substrate of OCT1) was explored. The minor effects of OCT1 on the pharmacokinetics and tissue accumulation suggested that the Oct1-mediated transport of oxaliplatin is not sufficiently high to remarkably affect its disposition in vivo. The increased hepatic platinum levels after multiple dosing in Oct1+/+ mice did not result in obvious liver toxicity, indicating that the hepatic toxicity of oxaliplatin observed in humans was not observed under the conditions used in this study and a clear role for Oct1 in oxaliplatin-induced hepatotoxicity could not be established.

The novel organic cation transporters (OCTNs) are bifunctional and bidirectional tranporters that are highly expressed at the apical membrane in renal tubular epithelia, and are thought to contribute to both the renal salvage of nutrients and to the active tubular secretion of xenobiotics. We used a genotype-to-phenotype approach to study the importance of OCTN1 (encoded by SLC22A4) and OCTN2 (encoded by SLC22A5) in the disposition of drugs and endobiotics. Resequencing of the coding region of these genes in a large ethnically-diverse sample of human DNA revealed six amino acid-altering nucleotide substitutions in SLC22A4 and eight in SLC22A5. When expressed heterologously, several rare variants of OCTN1 (D165G, R282X) were found to result in complete loss of transport function. Rare OCTN2 variants showed functional differences from the reference protein, including reduced function (V481F) and substrate selectivity differences (Y449D). Functionally significant single-nucleotide polymorphisms (SNPs) in OCTN1 (L503F) and OCTN2 (-207G>C and F17L) were also discovered. These common SNPs were tested for functional significance in vivo by recruiting subjects based on genotype at these positions, and testing for quantitative differences between genotype groups in the pharmacokinetics of gabapentin (an OCTN1 substrate) and carnitine (a preferred substrate of OCTN2). OCTN1 genotype was found to be a significant predictor of gabapentin renal clearance, with OCTN1 reference homozygotes showing net active secretion of gabapentin, but L503F homozygotes showing almost no active secretion of this drug. This finding adds the OCTN1-L503F polymorphism to a small list of SNPs known to influence drug disposition. OCTN genotype did not affect carnitine disposition or lipid metabolism. A meta-analysis of protein-altering variants of membrane transporters revealed that, as exemplified by the OCTNs, rare variants are more likely to affect global biochemical function; however, variants with substrate-specific effects (such as the OCTN1-L503F variant) may be relatively common. The results of this research support the hypothesis that OCTN transporters are important determinants of active drug secretion in the kidney, and that prediction of renal clearance of drugs may be improved by renal transporter genotype information. This research may help to inform future pharmacogenetic studies of the OCTN transporters, and renal drug transporters generally.

Members of the Solute Carrier (SLC) 22 family of transporters have been studied extensively due to their well-known role in drug excretion and disposition. However, numerous lines of evidence suggest that these transporters are likely to possess previously unrecognized roles in basal physiological processes. These studies were undertaken to better delineate the potential physiological importance of 2 organic anion transporters (OATs) expressed on the apical membrane of the apical membrane of renal tubule cells, SLC22A11 (OAT4) and SLC22A12 (URAT1), the impact of genetic variants of these two transporters, and to create a humanized mouse model suitable for an additional physiological and drug disposition studies of another SLC22 transporter, SLC22A1 (OCT1).

Of the 9 OAT4 nonsynonymous variants studied, the L29P, R48Stop, and H469R variants all displayed negligible uptake of 3 canonical, endogenous substrates (estrone sulfate, ochratoxin A, and uric acid) likely due to the absence of plasma membrane protein localization. Extending this research to URAT1, 4 nonsynonymous variants, G65W, I131V, W277R, and R342H, as well as a C-terminal frameshift variant T542LysfX13, all displayed some reduction in uptake of uric acid, with the T542LysfX13 reduction likely due to the loss of a protein-binding motif and resultant plasma membrane localization. Collectively, the presence of these variants provides mechanistic reasons for variability in reabsorption of solutes from the urine in the human population, potentially affecting metabolite homeostasis as well as drug pharmacokinetics.

Comparisons of the evolutionary, genetic, and molecular features of OAT4 and URAT1 demonstrated that both are under general purifying selection and that their evolution has diverged considerably within the mammalian lineage, with numerous species lacking clear orthologs for either transporter. Likewise, expression of OAT4 was also found in the epididymis, an embryonically related tissue to the kidney but involved in the maturation of spermatozoa. In addition, profiling of substrate preferences for these two transporters showed considerably divergent sets of interacting molecules, including the uptake of AMP and ADP by URAT1 and an apparent binding site size restriction difference. These results suggest that these transporters, while evolutionarily related, have diverged to accommodate specialized physiological roles in a subset of mammalian lineage.

Finally, a mouse model expressing human OCT1 in the liver was created using the mouse albumin core promoter and distal enhancer to drive liver-specific expression. Absolute OCT1 transgene expression levels were 4 times higher in the transgenic line compared to the normal human liver and 20 times higher than any other mouse tissue. While expression was not restricted to the liver as anticipated, this model can aid in the assessment of drug substrates of OCT1 and provide a flexible model to evaluate the physiological repercussions of human OCT1 in an intact animal model.

This work demonstrates that while these transporters are important from a drug development standpoint, they also may be involved in specialized physiological processes, which may be modulated by naturally occurring genetic variants. Additional studies are required to better understand the full impact of these transporters on human physiology and the resultant impact on human health.

Membrane transport proteins of the solute carrier (SLC) and ATP-binding cassette (ABC) superfamilies have been found to play essential roles in the absorption, distribution and elimination of drugs. However, much of the pharmacologic research on transporters has focused primarily on the intestine, liver and kidney. Here we focus on transporters in the blood-brain barrier (BBB), a complex barrier that limits penetration of most molecules from the blood into the brain. Research on transporters in the BBB has historically been centered on the ABC transporters that prevent drug entry into the brain, but recent advances suggest that SLC transporters may play an important role in mediating the uptake of many pharmacologic agents into the central nervous system (CNS). The goal of this dissertation research was to understand the role and function of several SLC transporters in the BBB, including the amino acid transporter LAT1, the organic cation transporter MATE1, the amine transporters OCT1, OCT3 and SERT, and the organic anion transporters OATP1A2 and OATP2B1. We performed inhibition and substrate screens using stably-transfected cell lines against a library of CNS-active drugs. We were able to identify four novel, structurally-diverse inhibitors of LAT1 and developed a rat perfusion model to test LAT1 substrates for in vivo relevance. For MATE1, we found that the majority of compounds tested from our library were inhibitors of MATE1, and identified 15 novel substrates, suggesting that MATE1 may be involved in the disposition of these drugs. Since organic anion transporting polypeptides are known to play a role in the influx of molecules into the brain, we performed substrate screens and identified 24 novel substrates of OATP1A2, most of which are cationic drugs. About one-third of the OATP1A2 substrates were also substrates of its rodent ortholog, rat Oatp2. Finally, we found that at high concentrations, metformin is able to inhibit the uptake of two CNS active monoamines, histamine and serotonin, by OCT1, OCT3 and SERT. Though further studies are clearly needed, we posit that reduced absorption of the two monoamines as a result of metformin’s effects on OCT1, OCT3 and SERT may contribute to the gastrointestinal side effects associated with metformin use. The research presented here has important implications for CNS drug delivery, as our results have expanded the chemical space, particularly the known substrates, of several transporters expressed in the BBB.

Drug metabolizing enzymes and membrane transporters work together in the absorption and disposition of drugs. Although the effects of ethnicity on drug metabolism are well studied, less is known about the ethnic differences in disposition of drugs that are substrates of membrane transporters. Organic anion transporters (OATs) are members of the SLC22 drug transporter family. OATs typically transport a wide variety of endogenous and xenobiotic organic anions. The goals of this dissertation were to determine the ethnic differences in allele frequencies of OAT2 (SLC22A7) and OAT3 (SLC22A8) variants and to determine if genetic variants of OAT2 and OAT3 impact the ability of these transporters to interact with drugs and endogenous substances. We showed that OAT2 is a novel, bidirectional, facilitative plasma membrane transporter for cGMP and other guanine nucleotides. cGMP exhibited a 50- to 100-fold enhanced uptake in OAT2 transfected cells over empty vector transfected cells. We observed that acyclovir, a guanine-like antiviral agent, was also an excellent substrate of OAT2. We sequenced the coding region of OAT2 in a collection of 272 DNA samples from an ethnically diverse cohort. Six rare non-synonymous variants were identified in the 272 DNA samples, suggesting that OAT2 is under strong negative selective pressure. Only Thr110Ile was polymorphic, with an allele frequency of 2.3% in African Americans. Acyclovir exhibited a significantly reduced uptake and Vmax in cells expressing Thr110Ile, in comparison to cells expressing the reference OAT2. OAT3, highly expressed in the basolateral membrane of renal proximal tubules, plays a significant role in the clearance of methotrexate, an anti-cancer and anti-inflammatory drug. We characterized the impact of OAT3 and its genetic variants on the transport of methotrexate and its metabolite, 7-hydroxymethotrexate. We showed that several OAT3 protein-altering variants exhibit significant differences in methotrexate transport compared to OAT3 reference in cellular assays. Genetic variants of OAT3 may contribute to inter-individual variation in methotrexate elimination and toxicity. These studies have led to an enhanced understanding of the role of OAT2, OAT3, and their genetic variants. The knowledge gained in this dissertation research will inform future clinical studies aimed at optimizing drug therapies in ethnically diverse populations.

Membrane transporters are widely expressed throughout the body, transporting nutrients, metabolites, toxins, and drugs across cellular membranes. In drug development, transporters are clinically important for drug absorption and disposition, and play significant roles in pharmacokinetics and treatment response. In addition, there is a growing interest in physiologic roles of transporters as polymorphisms in many drug transporters have been significantly associated with human diseases. Metformin is widely used as first-line treatment of type 2 diabetes. It exists primarily as a hydrophilic cation at physiological pHs, and therefore, membrane transporters are involved in its intestinal absorption, tissues distribution, and renal elimination. Multiple organic cation transporters play roles in the pharmacokinetics of metformin, and many of them play important roles in its pharmacological response, as mediators of metformin entry into target tissues. Here, we investigated transporters of metformin, with an emphasis on the pharmacological and biological roles of transporters in the intestine and liver. We characterized the human thiamine transporter (THTR-2; SLC19A3), which plays a major role in the intestinal absorption of thiamine (Vitamin B1). We found that human THTR-2 is capable of transporting other compounds including the drugs metformin and famotidine, and the neurotoxin MPP+. Other chemical agents including phenformin, chloroquine, verapamil, famotidine, amprolium, and pyrithiamine were inhibitors of hTHTR-2 mediated uptake of both thiamine and metformin. Interestingly, previous studies from our laboratory and others identified thiamine as an endogenous substrate for several organic cation transporters including OCT1, OCT2, and MATE1. In our second study, we focused on the endogenous role of OCT1, which is the major hepatic transporter for metformin. Through extensive experiments in Oct1 knockout mice, our data support the notion that hepatic thiamine deficiency is the underlying mechanism for the clinical phenotypes associated with reduced OCT1 function, particularly elevated LDL- cholesterol and total cholesterol levels. Our data suggest that reduced OCT1-mediated thiamine uptake in the liver leads to reduced levels of thiamine pyrophosphate, TPP, and a decreased activity of key TPP-dependent enzymes. As a result, there is a shift from glucose to fatty acid oxidation in the liver, which disrupts key metabolic flux pathways. In parallel, through analysis of published genomewide association studies, we show that reduced function OCT1 polymorphisms are associated with several metabolic traits in humans. Taken together, our studies suggest that changes in OCT1 activity modulate the disposition of thiamine and its active metabolite, TPP, which results in alterations in hepatic energy metabolism, ultimately affecting total body energy homeostasis. Finally, we developed a comparative structural model of OCT1 that discriminates ligands from non-ligands and is the model together with an in vitro high-throughput screening (HTS) assay to screen a drug library to explore the molecular properties of drugs interacting with OCT1. To extend the in silico methods in predicting endogenous metabolites that interact with OCT1, we conducted a virtual screen of 29,332 human metabolites against the structure-based model and an SAR model generated from the HTS data. In conclusion, our findings have important implications for understanding metformin absorption and disposition, and have expanded our understanding of the physiologic and pathophysiologic roles of membrane transporters.

Metformin is used first line for treatment of type 2 diabetes (T2D) and is one of the most frequently prescribed drugs worldwide. As the global incidence of T2D rapidly increases, the low cost of metformin makes this treatment option particularly attractive in developing nations. Understanding metformin’s efficacy in different patient populations with diverse genetic backgrounds will be critical in managing this deleterious metabolic disorder. The major goal of this dissertation research was to use novel, quantitative approaches to elucidate genetic and non-genetic components that predict metformin disposition and glycemic response. As a first goal, the role of transcription factor variants on metformin pharmacokinetics and pharmacodynamics was investigated. From this analysis, five variants in SP1 were significantly associated with changes in treatment HbA1c (p < 0.01) and metformin secretory clearance (p < 0.05). Genetic variants in transcription factors PPAR-alpha and HNF4-alpha were significantly associated with HbA1c change only, but were not significantly associated with pharmacokinetics. A plausible biological mechanism by which genetic variants affected the pharmacological variation of metformin was determined using gene expression levels linked to genetic variants (eQTLs). The focus was on transporter expression. From this study, we discovered that genomic regions proximal to metformin transporters were linked to expression levels of SLC47A1, SLC22A3, and SLC22A2, with a potential transcription factor-binding hypothesis for SP1. We also found variants in transcription factor HNF4-alpha were the most influential trans-eQTLs, accounting for expression level variation in both SLC47A1 and SLC22A1. Finally, we developed a mathematical model to quantify disease progression on metformin therapy using HbA1c data with the goal of explaining long-term HbA1c variability through the investigation of genetic, demographic, and clinical factors. From this analysis, we found two SNPs in CSMD1 (rs2617102, rs2954625) and one SNP in SLC22A2 (rs316009) as significantly influencing the long-term variance in HbA1c. Overall, this dissertation research enhances our current knowledge of the pharmacogenetic landscape by expanding the set of pharmacologically relevant genes and providing a pharmacokinetic and biological basis for some of these genes. Future research will continue to focus on replication and uncovering the mechanism driving the pharmacological genes highlighted here.

ABSTRACT

Renal transporters are primary modulators of drug disposition and elimination. The goals of this dissertation are: 1) to understand the role of renal transporters and genetic variation in renal transporters in disease through epidemiological studies and genetic association studies; 2) to characterize the role of transporters and assess the contribution of genetic variation in drug transport and cytotoxicity through functional experimental studies, 3) to identify and predict selective inhibitors of OAT1 and OAT3 through high throughput screening and computational modeling.

Our epidemiological study of tenofovir associated renal insufficiency demonstrates that tenofovir is associated with mild decline in renal function and that HIV therapy, in general, may improve renal function in women with pre-existing renal impairment. Functional studies demonstrate that SLC22A6 is a primary transporter for tenofovir and adefovir uptake in the renal proximal tubule. We found that rare variants in SLC22A6 may play a role in differential transport and cytotoxicity of tenofovir and adefovir. A genetic association study using DNA from 565 children with pediatric leukemia on methotrexate therapy demonstrated that in 8 candidate transporter genes chosen, 11 single nucleotide polymorphisms (SNPs) out of 44 SNPs in ABCC1, ABCC2, ABCC4, ABCG2, and SLC22A8 were significantly associated with changes in glomerular filtration rate(GFR) post-methotrexate. Haplotype blocks of the ABCG2 were found to be significantly associated with changes in GFR after methotrexate treatment. Findings from our chemo-informatics screening study suggest that SLC22A6 and SLC22A8 molecular interactors vary in structure and that small changes in electrostatics and steric effects can drastically change the inhibition activity of SLC22A6 and SLC22A8. A number of non-selective and selective inhibitors were identified. SAR studies identified several chemical descriptors unique to OAT1 and OAT3 interacting molecules.

This dissertation provides a unique look at drug-induced nephrotoxicity from the vantage of pharmacogenetic variation in the SLC22A6 and SLC22A8 transporters. It sets the stage for investigations where other genes involved in the nephrotoxicity pathways of these drugs may be investigated. It is our hope that insights from this dissertation can be further extended to other transporters in the SLC22 family and may inform clinical decisions in the future.