Hong Shen

Monitoring endogenous biomarkers is increasingly used to evaluate transporter‐mediated drug–drug interactions (DDIs) in early drug development and may be applied to elucidate changes in transporter activity in disease. 4‐pyridoxic acid (PDA) has been identified as the most sensitive plasma endogenous biomarker of renal organic anion transporters (OAT1/3). Increase in PDA baseline concentrations was observed after administration of probenecid, a strong clinical inhibitor of OAT1/3 and also in patients with chronic kidney disease (CKD). The aim of this study was to develop and verify a physiologically‐based pharmacokinetic (PBPK) model of PDA, to predict the magnitude of probenecid DDI and predict the CKD‐related changes in PDA baseline. The PBPK model for PDA was first developed in healthy population, building on from previous population pharmacokinetic modeling, and incorporating a mechanistic kidney model to consider OAT1/3‐mediated renal secretion. Probenecid PBPK model was adapted from the Simcyp database and re‐verified to capture its dose‐dependent pharmacokinetics (n = 9 studies). The PBPK model successfully predicted the PDA plasma concentrations, area under the curve, and renal clearance in healthy subjects at baseline and after single/multiple probenecid doses. Prospective simulations in severe CKD predicted successfully the increase in PDA plasma concentration relative to healthy (within 2‐fold of observed data) after accounting for 60% increase in fraction unbound in plasma and additional 50% decline in OAT1/3 activity beyond the decrease in glomerular filtration rate. The verified PDA PBPK model supports future robust evaluation of OAT1/3 DDI in drug development and increases our confidence in predicting exposure and renal secretion in patients with CKD.

Coproporphyrins I and III (CP-I and CP-III) are established as substrates of hepatic sinusoidal organic anion transporting polypeptides (Oatps), multidrug resistance-associated protein 3 (Mrp3), and canalicular transporter Mrp2. We aimed to evaluate the potential of plasma CP-I and CP-III concentrations to indicate functional changes in hepatic transporters in nonalcoholic stea-tohepatitis (NASH) rodent models using modeling and simulation approach. Mechanistic copro-porphyrins (CP-I and CP-III) models in rats and mice were constructed based on metabolism, transport, and elimination pathways in rodents. To parameterize the rat mechanistic model, we measured CP concentrations in plasma, liver, bile, and urine in healthy control Sprague Dawley rats. The mouse mechanistic model was parameterized using published data. The rat and mouse CP models described the steady-state levels of CP-I and CP-III in plasma, liver, and bile. The model recapitulated the observed high plasma CP-III / CP-1 ratio (9- and 3-fold in rats and mice, respec-tively) and elimination pathways of CP-I (>90% via biliary secretion) and CP-III (equal via bile and urine). Rat CP model was further validated with the published rat fistula model. The simulated increased plasma CP-I concentrations (4-fold), resulted from decreased Oatp activity (0.6-fold) and increased Mrp3 activity (3-fold) were comparable to those observed in bile-duct ligated NASH model. Sensitivity analysis revealed that 90% inhibition of Oatp and Mrp2 leads to a 4-fold and 2.5-fold increase in plasma CP-I levels, respectively. Additionally, we demonstrated the utility of the mechanistic CP models in testing hypothesis and understanding the mechanisms of the ob-served plasma CPs disposition in the Oatp knockout (KO) mice, bile-duct ligation NASH mice, and Mrp2 deficient TR- rats. Collectively, using a mechanistic model, we conclude that CP-I is a robust and predictable marker for assessing change in the activity of Oatps and Mrps in animal models. Our analysis suggests that efflux transporters (Mrp2 and Mrp3) can play an important role in the disposition of CP-I along with Oatp transporters in rodents.

Purpose The differences between intestinal and systemic (hepatic and renal) P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) roles in drug disposition are difficult to define. Accordingly, we characterized Encequidar (ECD) as an intestinal P-gp and BCRP specific inhibitor to evaluate their role in drug disposition. Methods We assessed the in vitro and in vivo inhibition potential of ECD towards human and animal P-gp and BCRP. Results ECD is a potent inhibitor with a high degree of selectivity in inhibiting human P-gp (hP-gp) over human BCRP (hBCRP) (IC₅₀s of 0.0058 ± 0.0006 vs. > 10 µM, respectively). In contrast, ECD is a potent inhibitor of rat and cynomolgus monkey BCRP (IC₅₀ ranged from 0.059 to 0.18 µM). While the AUC of IV paclitaxel (PTX) was significantly increased by elacridar (ELD) (P < 0.05) but not ECD in rats (15 mg/kg; PO) (2.55- vs. 0.93-fold), that of PO PTX was significantly elevated to a similar extent between the inhibitors (39.5- vs. 33.5-fold). Similarly, the AUC of PO sulfasalazine (SFZ) was dramatically increased by ELD and ECD (16.6- vs. 3.04-fold) although that of IV SFZ was not significantly affected by ELD and ECD in rats (1.18- vs. 1.06-fold). Finally, a comparable ECD-induced increase of the AUC of PO talinolol in cynomolgus monkeys was observed compared with ELD (2.14- vs. 2.12-fold). Conclusions ECD may allow an in-depth appraisal of the role of intestinal efflux transporter(s) in drug disposition in animals and humans through local intestinal drug interactions.

Madin-Darby canine kidney (MDCK) cells are widely used to study epithelial cell functionality. Their low endogenous drug transporter protein levels make them an amenable system to investigate transepithelial permeation and drug transporter protein activity after their transfection. MDCK cells display diverse phenotypic traits, and as such, laboratory-to-laboratory variability in drug permeability assessments is observed. Consequently, in vitro-in vivo extrapolation (IVIVE) approaches using permeability and/or transporter activity data require calibration. A comprehensive proteomic quantification of 11 filter-grown parental or mock-transfected MDCK monolayers from 8 different pharmaceutical laboratories using the total protein approach (TPA) is provided. The TPA enables estimations of key morphometric parameters such as monolayer cellularity and volume. Overall, metabolic liability to xenobiotics is likely to be limited for MDCK cells due to the low expression of required enzymes. SLC16A1 (MCT1) was the highest abundant SLC transporter linked to xenobiotic activity, while ABCC4 (MRP4) was the highest abundant ABC transporter. Our data supports existing findings that claudin-2 levels may be linked to tight junction modulation, thus impacting trans-epithelial resistance. This unique database provides data on more than 8000 protein copy numbers and concentrations, thus allowing an in-depth appraisal of the control monolayers used in each laboratory.

Advancement of endogenous biomarkers for drug transporters as a tool for assessing drug-drug interactions (DDIs) depends on initial identification of biomarker candidates and relies heavily on biomarker validation and its response to reference inhibitors in vivo. To identify endogenous biomarkers of breast cancer resistance protein (BCRP), we applied metabolomic approaches to profile plasma from Bcrp−/−, Mdr1a/1b−/−, and Bcrp/Mdr1a/1b−/− mice. Approximately 130 metabolites were significantly altered in Bcrp and P-gp knockout mice, indicating numerous metabolite-transporter interactions. We focused on BCRP specific substrates and identified riboflavin, which was significantly elevated in the plasma of Bcrp single- and Bcrp/P-gp double- but not P-gp single-knockout mice. Dual BCRP/P-gp inhibitor elacridar caused a dose-dependent increase of the area under the plasma concentration-time curve (AUC) of riboflavin in mice (1.51- and 1.93-fold increases by 30 and 150 mg/kg elacridar, respectively). In 3 cynomolgus monkeys, we observed approximately 1.7-fold increases in the riboflavin concentrations caused by ML753286 (10 mg/kg), which correlated well with the increase of sulfasalazine, a known BCRP probe in monkeys. However, the BCRP inhibitor had no effect on isobutyryl carnitine, arginine or 2-arachidonoyl glycerol levels. Additionally, clinical studies on healthy volunteers indicated low intrasubject and inter-meal variability of plasma riboflavin concentrations. In vitro experiments using membrane vesicles demonstrated riboflavin as a select substrate of monkey and human BCRP over P-gp. Collectively, this proof-of-principle study indicates that riboflavin is a suitable endogenous probe for BCRP activity in mice and monkeys and future investigation of riboflavin as a blood-based biomarker of human BCRP is warranted. Significance Statement Our results identified riboflavin as an endogenous biomarker candidate of BCRP. Its selectivity, sensitivity, and predictivity regarding BCRP inhibition have been explored. The findings of this study highlight riboflavin as an informative BCRP plasma biomarker in animal models. The utility of this biomarker requires further validation by evaluating the effects of BCRP inhibitors of different potencies on riboflavin plasma concentrations in humans. Ultimately, riboflavin may shed light on the risk assessment of BCRP DDIs in early clinical trials.

One challenge in central nervous system (CNS) drug discovery has been ensuring the blood–brain barrier (BBB) penetration of compounds at an efficacious concentration that provides suitable safety margins for clinical investigation. Research providing for the accurate prediction of brain penetration of compounds during preclinical discovery is important to a CNS program. In the BBB, P‐glycoprotein (P‐gp) (ABCB1) and breast cancer resistance protein (BCRP) (ABCG2) transporters have been demonstrated to play a major role in the active efflux of endogenous compounds and xenobiotics out of the brain microvessel cells and back to the systemic circulation. In the past 10 years, there has been significant technological improvement in the sensitivity of quantitative proteomics methods, in vivo imaging, in vitro methods of organoid and microphysiological systems, as well as in silico quantitative physiological based pharmacokinetic and systems pharmacology models. Scientists continually leverage these advancements to interrogate the distribution of compounds in the CNS which may also show signals of substrate specificity of P‐gp and/or BCRP. These methods have shown promise toward predicting and quantifying the unbound concentration(s) within the brain relevant for efficacy or safety. In this review, the authors have summarized the in vivo, in vitro, and proteomics advancements toward understanding the contribution of P‐gp and/or BCRP in restricting the entry of compounds to the CNS of either healthy or special populations. Special emphasis has been provided on recent investigations on the application of a proteomics‐informed approach to predict steady‐state drug concentrations in the brain. Moreover, future perspectives regarding the role of these transporters in newer modalities are discussed.

Farnesoid X receptor (FXR) is a nuclear receptor known to markedly alter expression of major transporters and enzymes in liver. However, its effects toward OATP1B1 and OATP1B3 remain poorly characterized. Therefore, the present study was aimed at determining the effects of chenodeoxycholic acid (CDCA), a naturally occurring FXR agonist, on OATP1B expression in cynomolgus monkeys. Multiple administration of 50 and 100 mg/kg CDCA was first shown to significantly repress mRNA expression of SLCO1B1/3 approximately 60% to 80% in monkey livers. It also suppressed cytochrome P450 (CYP)7A1-mRNA and induced OSTα/β-mRNA, which are well known targets of FXR and determinants of bile acid homeostasis. CDCA concomitantly decreased OATP1B protein abundance by approximately 60% in monkey liver. In contrast, multiple doses of 15 mg/kg rifampin (RIF), a pregnane X receptor (PXR) agonist, had no effect on hepatic OATP1B protein although it induced the intestinal P-gp and MR2 proteins by ~2-fold. Moreover, multiple doses of CDCA resulted in a steady ~2- to 10-fold increase of the OATP1B biomarkers coproporphyrins (CPs) in the plasma samples collected prior to each CDCA dose. Additionally, 3.4- to 11.2-fold increases of CPI and CPIII AUCs were observed after multiple administrations compared to the single dose and vehicle administration dosing groups. Taken together, these data suggest that CDCA represses the expression of OATP1B1 and OATP1B3 in monkeys. Further investigation of OATP1B down-regulation by FXR in humans is warranted, as such down-regulation effects may be involved in bile acid hemostasis and potential drug interactions in man. Significance Statement Using gene expression and proteomics tools, as well as endogenous biomarker data, for the first time, we have demonstrated that OATP1B expression was suppressed and its activity was reduced in the cynomolgus monkeys following oral administration of 50 and 100 mg/kg/day CDCA, a FXR agonist, for 8 days. These results lead to a better understanding of OATP1B down-regulation by CDCA and its role on bile acid and drug disposition.

Membrane transporters have been recognized as one of the key determinants of pharmacokinetics and are also known to affect the efficacy and toxicity of drugs. Both qualitatively and quantitatively, however, transporter studies conducted using human in vitro systems have not always been predictive. Consequently, researchers have utilized cynomolgus monkeys as a model to study drug transporters and anticipate their effects in humans. Burgeoning reports of data in the last few years necessitates a comprehensive review on the topic of drug transporters in cynomolgus monkeys that includes cell-based tools, sequence homology, tissue expression, in vitro studies, in vivo studies, and in vitro-to-in vivo extrapolation (IVIVE). This review highlights the state-of-the-art applications of monkey transporter models to support the evaluation of transporter-mediated drug-drug interactions, clearance predictions, and endogenous transporter biomarker identification and validation. The data demonstrate that cynomolgus monkey transporter models, when used appropriately, can be an invaluable tool to support drug discovery and development processes. Most importantly, they provide an early IVIVE assessment which provides additional context to human in vitro data. Additionally, comprehending species similarities and differences in transporter tissue expression and activity is crucial when translating monkey data to humans. The challenges and limitations when applying such models to inform decision-making must also be considered. Significance Statement This paper presents a comprehensive review of currently available published reports describing cynomolgus monkey transporter models. The data indicate that cynomolgus monkeys provide mechanistic insight regarding the role of intestinal, hepatic, and renal transporters in drug and biomarker disposition and drug interactions. It is concluded that the data generated with cynomolgus monkey models provide mechanistic insight regarding transporter-mediated absorption and disposition, as well as human clearance prediction, drug-drug interaction assessment, and endogenous biomarker development related to drug transporters.