Incorporating Uremic Solute-mediated Inhibition of OAT1/3 Improves PBPK Prediction of Tenofovir Renal and Systemic Disposition in Patients with Severe Kidney Disease.

BACKGROUND: Dose modification of renally secreted drugs in patients with chronic kidney disease (CKD) has relied on serum creatinine concentration as a biomarker to estimate glomerular filtration (GFR) under the assumption that filtration and secretion decline in parallel. A discrepancy between actual renal clearance and predicted renal clearance based on GFR alone is observed in severe CKD patients with tenofovir, a compound secreted by renal OAT1/3. Uremic solutes that inhibit OAT1/3 may play a role in this divergence. METHODS: To examine the impact of transporter inhibition by uremic solutes on tenofovir renal clearance, we determined the inhibitory potential of uremic solutes hippuric acid, indoxyl sulfate, and p-cresol sulfate. The inhibition parameters (IC50) were incorporated into a previously validated mechanistic kidney model; simulated renal clearance and plasma PK profile were compared to data from clinical studies. RESULTS: Without the incorporation of uremic solute inhibition, the PBPK model failed to capture the observed data with an absolute average fold error (AAFE) > 2. However, when the inhibition of renal uptake transporters and uptake transporters in the slow distribution tissues were included, the AAFE value was within the pre-defined twofold model acceptance criterion, demonstrating successful model extrapolation to CKD patients. CONCLUSION: A PBPK model that incorporates inhibition by uremic solutes has potential to better predict renal clearance and systemic disposition of secreted drugs in patients with CKD. Ongoing research is warranted to determine if the model can be expanded to include other OAT1/3 substrate drugs and to evaluate how these findings can be translated to clinical guidance for drug selection and dose optimization in patients with CKD.

Recent advances in the ontogeny of drug disposition.

Developmental changes that occur throughout childhood have long been known to impact drug disposition. However, pharmacokinetic studies in the paediatric population have historically been limited due to ethical concerns arising from incorporating children into clinical trials. As such, much of the early work in the field of developmental pharmacology was reliant on difficult-to-interpret in vitro and in vivo animal studies. Over the last 2 decades, our understanding of the mechanistic processes underlying age-related changes in drug disposition has advanced considerably. Progress has largely been driven by technological advances in mass spectrometry-based methods for quantifying proteins implicated in drug disposition, and in silico tools that leverage these data to predict age-related changes in pharmacokinetics. This review summarizes our current understanding of the impact of childhood development on drug disposition, particularly focusing on research of the past 20 years, but also highlighting select examples of earlier foundational research. Equally important to the studies reviewed herein are the areas that we cannot currently describe due to the lack of research evidence; these gaps provide a map of drug disposition pathways for which developmental trends still need to be characterized.

Bridging the gap between in silico and in vivo by modeling opioid disposition in a kidney proximal tubule microphysiological system.

Opioid overdose, dependence, and addiction are a major public health crisis. Patients with chronic kidney disease (CKD) are at high risk of opioid overdose, therefore novel methods that provide accurate prediction of renal clearance (CLr) and systemic disposition of opioids in CKD patients can facilitate the optimization of therapeutic regimens. The present study aimed to predict renal clearance and systemic disposition of morphine and its active metabolite morphine-6-glucuronide (M6G) in CKD patients using a vascularized human proximal tubule microphysiological system (VPT-MPS) coupled with a parent-metabolite full body physiologically-based pharmacokinetic (PBPK) model. The VPT-MPS, populated with a human umbilical vein endothelial cell (HUVEC) channel and an adjacent human primary proximal tubular epithelial cells (PTEC) channel, successfully demonstrated secretory transport of morphine and M6G from the HUVEC channel into the PTEC channel. The in vitro data generated by VPT-MPS were incorporated into a mechanistic kidney model and parent-metabolite full body PBPK model to predict CLr and systemic disposition of morphine and M6G, resulting in successful prediction of CLr and the plasma concentration-time profiles in both healthy subjects and CKD patients. A microphysiological system together with mathematical modeling successfully predicted renal clearance and systemic disposition of opioids in CKD patients and healthy subjects.

An Improved Vascularized, Dual-Channel Microphysiological System Facilitates Modeling of Proximal Tubular Solute Secretion.

A vascularized human proximal tubule model in a dual-channel microphysiological system (VPT-MPS) was developed, representing an advance over previous, single-cell-type kidney microphysiological systems. Human proximal tubule epithelial cells (PTECs) and human umbilical vein endothelial cells (HUVECs) were cocultured in side-by-side channels. Over 24 h of coculturing, PTECs maintained polarized expression of Na+/K+ ATPase, tight junctions (ZO-1), and OAT1. HUVECs showed the absence of ZO-1 but expressed endothelial cell marker (CD-31). In time-lapse imaging studies, fluorescein isothiocyanate (FITC)-dextran passed freely from the HUVEC vessel into the supporting extracellular matrix, confirming the leakiness of the endothelium (at 80 min, matrix/intravessel fluorescence ratio = 0.2). Dextran-associated fluorescence accumulated in the matrix adjacent to the basolateral aspect of the PTEC tubule with minimal passage of the compound into the tubule lumen observed (at 80 min, tubule lumen/matrix fluorescence ratio = 0.01). This demonstrates that the proximal tubule compartment is the rate-limiting step in the secretion of compounds in VPT-MPS. In kinetic studies with radiolabeled markers, p-aminohippuric acid (PAH) exhibited greater output into the tubule lumen than did paracellular markers mannitol and FITC-dextran (tubule outflow/vessel outflow concentration ratio of 7.7% vs 0.5 and 0.4%, respectively). A trend toward reduced PAH secretion by 45% was observed upon coadministration of probenecid. This signifies functional expression of renal transporters in PTECs that normally mediate the renal secretion of PAH. The VPT-MPS holds the promise of providing an in vitro platform for evaluating the renal secretion of new drug candidates and investigating the dysregulation of tubular drug secretion in chronic kidney disease.

Reevaluating the role of megalin in renal vitamin D homeostasis using a human cell-derived microphysiological system

The role of megalin in the regulation of renal vitamin D homeostasis has previously been evaluated in megalin-knockout mice and rat proximal tubule epithelial cells. We revisited these hypotheses that were previously tested solely in rodent models, this time using a 3-dimensional proximal tubule microphysiological system incorporating primary human proximal tubule epithelial cells. Using this human cell-derived model, we confirmed that 25OHD3 is transported into the human proximal tubule epithelium via megalin-mediated endocytosis while bound to vitamin D binding protein. Building upon these findings, we then evaluated the role of megalin in modulating the cellular uptake and biological activity of 1α,25(OH)2D3. Inhibition of megalin function decreased the 1α,25(OH)2D3-mediated induction of both cytochrome P450 24A1 protein levels and 24-hydroxylation activity following perfusion with vitamin D binding protein and 1α,25(OH)2D3. The potential for reciprocal effects from 1α,25(OH)2D3 on megalin expression were also tested. Contrary to previously published observations from rat proximal tubule epithelial cells, 1α,25(OH)2D3 did not induce megalin gene expression, thus highlighting the potential for meaningful interspecies differences in the homeostatic regulation of megalin in rodents and humans. These findings challenge a recently promoted hypothesis, predicated on the rodent cell data, that attempts to connect 1α,25(OH)2D3-mediated regulation of renal megalin expression and the pathology of chronic kidney disease in humans. In addition to providing specific insights related to the importance of renal megalin in vitamin D homeostasis, these results constitute a proof-of-concept that human-derived microphysio­logical systems are a suitable replacement for animal models for quantitative pharmacology and physiology research.

Polymorphic Human Sulfotransferase 2A1 Mediates the Formation of 25-Hydroxyvitamin D3-3-O-Sulfate, a Major Circulating Vitamin D Metabolite in Humans.

Metabolism of 25-hydroxyvitamin D3 (25OHD3) plays a central role in regulating the biologic effects of vitamin D in the body. Although cytochrome P450-dependent hydroxylation of 25OHD3 has been extensively investigated, limited information is available on the conjugation of 25OHD3 In this study, we report that 25OHD3 is selectively conjugated to 25OHD3-3-O-sulfate by human sulfotransferase 2A1 (SULT2A1) and that the liver is a primary site of metabolite formation. At a low (50 nM) concentration of 25OHD3, 25OHD3-3-O-sulfate was the most abundant metabolite, with an intrinsic clearance approximately 8-fold higher than the next most efficient metabolic route. In addition, 25OHD3 sulfonation was not inducible by the potent human pregnane X receptor agonist, rifampicin. The 25OHD3 sulfonation rates in a bank of 258 different human liver cytosols were highly variable but correlated with the rates of dehydroepiandrosterone sulfonation. Further analysis revealed a significant association between a common single nucleotide variant within intron 1 of SULT2A1 (rs296361; minor allele frequency = 15% in whites) and liver cytosolic SULT2A1 content as well as 25OHD3-3-O-sulfate formation rate, suggesting that variation in the SULT2A1 gene contributes importantly to interindividual differences in vitamin D homeostasis. Finally, 25OHD3-3-O-sulfate exhibited high affinity for the vitamin D binding protein and was detectable in human plasma and bile but not in urine samples. Thus, circulating concentrations of 25OHD3-3-O-sulfate appear to be protected from rapid renal elimination, raising the possibility that the sulfate metabolite may serve as a reservoir of 25OHD3 in vivo, and contribute indirectly to the biologic effects of vitamin D.

Human liver-kidney model elucidates the mechanisms of aristolochic acid nephrotoxicity.

Environmental exposures pose a significant threat to human health. However, it is often difficult to study toxicological mechanisms in human subjects due to ethical concerns. Plant-derived aristolochic acids are among the most potent nephrotoxins and carcinogens discovered to date, yet the mechanism of bioactivation in humans remains poorly understood. Microphysiological systems (organs-on-chips) provide an approach to examining the complex, species-specific toxicological effects of pharmaceutical and environmental chemicals using human cells. We microfluidically linked a kidney-on-a-chip with a liver-on-a-chip to determine the mechanisms of bioactivation and transport of aristolochic acid I (AA-I), an established nephrotoxin and human carcinogen. We demonstrate that human hepatocyte-specific metabolism of AA-I substantially increases its cytotoxicity toward human kidney proximal tubular epithelial cells, including formation of aristolactam adducts and release of kidney injury biomarkers. Hepatic biotransformation of AA-I to a nephrotoxic metabolite involves nitroreduction, followed by sulfate conjugation. Here, we identify, in a human tissue-based system, that the sulfate conjugate of the hepatic NQO1-generated aristolactam product of AA-I (AL-I-NOSO3) is the nephrotoxic form of AA-I. This conjugate can be transported out of liver via MRP membrane transporters and then actively transported into kidney tissue via one or more organic anionic membrane transporters. This integrated microphysiological system provides an ex vivo approach for investigating organ-organ interactions, whereby the metabolism of a drug or other xenobiotic by one tissue may influence its toxicity toward another, and represents an experimental approach for studying chemical toxicity related to environmental and other toxic exposures.

Potent inhibition of human organic cation transporter 2 (hOCT2) by ß-carboline alkaloids.

1. Beta-carbolines are indole alkaloids with a wide range of pharmacological and toxicological activities. Beta-carbolines are structurally related to the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), a known substrate of organic cation transporters (OCTs). The goal of this study is to determine the interaction of ß-carbolines with human OCT1, 2, and 3 (SLC22A1-3). 2. Dose-dependent inhibition studies were performed for five commercially available ß-carbolines using a fluorescent substrate assay in HEK293 cells stably expressing hOCT1-3. The substrate potential was evaluated by uptake assays and the impact of active transport on cellular toxicity examined. 3. All tested ß-carbolines potently inhibited hOCT2 with IC50 values in the sub- or low micromolar range. Harmaline is the most potent hOCT2 inhibitor (IC50 = 0.50 ± 0.08 µM). hOCT1 and hOCT3 are less sensitive to ß-carboline inhibition. Harmaline, norharmanium, and 2,9-dimethyl-4,9-dihydro-3H-ß-carbolinium accumulated 2- to 7-fold higher in cells expressing hOCT1-3. HEK293 cells expressing hOCT1-3 were 6.5- to 13-fold more sensitive to harmane and norharmanium toxicity. 4. Our data support a significant role of hOCT1-3 in tissue uptake and disposition of ß-carbolines. Importantly, the potent inhibition of hOCT2 by ß-carbolines also raises the concern of potential drug interactions between naturally occurring bioactive alkaloids and drugs eliminated by hOCT2.

Development of a microphysiological model of human kidney proximal tubule function.

The kidney proximal tubule is the primary site in the nephron for excretion of waste products through a combination of active uptake and secretory processes and is also a primary target of drug-induced nephrotoxicity. Here, we describe the development and functional characterization of a 3-dimensional flow-directed human kidney proximal tubule microphysiological system. The system replicates the polarity of the proximal tubule, expresses appropriate marker proteins, exhibits biochemical and synthetic activities, as well as secretory and reabsorptive processes associated with proximal tubule function in vivo. This microphysiological system can serve as an ideal platform for ex vivo modeling of renal drug clearance and drug-induced nephrotoxicity. Additionally, this novel system can be used for preclinical screening of new chemical compounds prior to initiating human clinical trials.