Stimulus-responsive mode is highly desirable for improving the precise monitoring and physiological efficacy of endogenous biomarkers (EB). However, its integrated application for visual detection and therapy is limited by inappropriate use of responsive triggers and poor delivery of EB signal-transducing agents, which remain challenging in simultaneous monitoring and noninvasive therapy of EB and EB-mediated pathological events. Target microRNA (miRNA) as controllable reaction triggers and DNAzyme as signal-transducing agent are proposed to develop target-stimulated multifunctional nanocabinets (MFNCs) for the visual tracking of both miRNA and miRNA-mediated anticancer events. The MFNCs, equipped with a target-discriminating sequence-incorporated DNAzyme motif, can specifically release therapeutic molecules through target-triggered conformational switches, accompanied by transduction signal output. Target detection and molecule release performance are recorded in parallel via reverse dual-signal feedback at the single-molecule level. In addition, the intrinsic thermal-replenishing of the MFNCs leads to tumor ablation without invasive exogenous aids. The system achieves visual target quantification, anticancer molecule real-time tracking, and tumor suppression in vivo and in vitro. This work proposes a new paradigm for precise visual exploration of EB or EB-mediated bio-events and provides a demonstration of efficacious all-in-one detection and therapy based on the target-triggered multifunctional nanosystem.

In recent years, various endogenous compounds have been proposed as putative biomarkers for the hepatic uptake transporters OATP1B1 and OATP1B3 that have the potential to predict transporter-mediated drug-drug interactions (DDIs). However, these compounds have often been identified from top-down strategies and have not been fully utilized as a substitute for traditional DDI studies. In an attempt to eliminate observer bias in biomarker selection, we applied a bottom-up, untargeted metabolomics screening approach in mice and found that plasma levels of the conjugated bile acid chenodeoxycholate-24-glucuronide (CDCA-24G) are particularly sensitive to deletion of the orthologous murine transporter Oatp1b2 (31-fold increase vs. wild type) or the entire Oatp1a/1b(-/-)cluster (83-fold increased), whereas the humanized transgenic overexpression of hepatic OATP1B1 or OATP1B3 resulted in the partial restoration of transport function. Validation studies with the OATP1B1/OATP1B3 inhibitors rifampin and paclitaxel in vitro as well as in mice and human subjects confirmed that CDCA-24G is a sensitive and rapid response biomarker to dose-dependent transporter inhibition. Collectively, our study confirmed the ability of CDCA-24G to serve as a sensitive and selective endogenous biomarker of OATP1B-type transport function and suggests a template for the future development of biomarkers for other clinically important xenobiotic transporters.

Glycochenodeoxycholate-3-sulfate (GCDCA-S) and chenodeoxycholate-24-glucuronide (CDCA-24G) are bile acid metabolites that potentially serve as endogenous biomarkers for drug-drug interactions mediated by the hepatic uptake transporters OATP1B1 and OATP1B3. We developed and validated a novel UHPLC-MS/MS method for the quantitative determination of GCDCA-S and CDCA-24G in mouse and human plasma with a lower limit of quantitation of 0.5 ng/mL. Chromatographic separation was achieved on an Accucore aQ column (50 mm × 2.1 mm, dp = 2.6 μm) maintained at 20 °C and a gradient mobile phase comprising 2 mM ammonium acetate in water and methanol. The extraction recoveries of GCDCA-S and CDCA-24G were >80 %, and linear (r2 > 0.99) calibration curves ranged 0.5-100 ng/mL (CDCA-24G and GCDCA-S in mouse plasma) or 0.5-1000 ng/mL (GCDCA-S in mouse plasma). Values for precision (CV < 11.6 %) and accuracy bias (10.9 %) of analyte-spiked quality control samples verified that water was an acceptable matrix to prepare calibrators. This method was successfully applied to establish baseline activity of OATP1B1/OATP1B3 in humans and mice and establish the in vivo effects of OATP1B1/OATP1B3 inhibitors rifampin and micafungin.

Comprehensive characterization of transporter mediated drug-drug interactions (DDIs) is important to formulate clinical management strategies and ensure the safe and effective use of concomitantly administered drugs. The potential of a drug to inhibit transporters is predicted by comparing the ratio of the relevant concentration (depending on the transporter) and the half maximum inhibitory concentration to a predefined \"cutoff\" value. If the ratio is greater than the cutoff value, modeling approaches such as physiologically based pharmacokinetic modeling or a clinical DDI trial may be recommended. Because false-positive (in vitro data suggest the potential for a DDI, whereas no significant DDI is observed in vivo) and false-negative (in vitro data does not suggest the potential for a DDI, whereas significant DDI is observed in vivo) outcomes have been observed, there is interest in exploring additional approaches to facilitate prediction of transporter-mediated DDIs. The idea of assessing changes in the concentration of endogenous biomarkers (which are substrates of clinically relevant transporters) to gain insight on the potential for a drug to inhibit transporter activity has received widespread attention. This brief report describes how endogenous biomarkers may help to expand the DDI assessment toolkit, highlights some current knowledge gaps, and outlines a conceptual framework that may complement the current paradigm of predicting the potential for transporter-mediated DDIs.

Rifampin has acute inhibitory and chronic inductive effects that can cause complex drug-drug interactions. Rifampin inhibits transporters including organic-anion-transporting polypeptide (OATP)1B and P-glycoprotein (P-gp), and induces enzymes and transporters including cytochrome P450 3A, UDP-glucuronosyltransferase (UGT)1A, and P-gp. This study aimed at separating inhibitory and inductive effects of rifampin on letermovir disposition and elimination (indicated for cytomegalovirus prophylaxis in hematopoietic stem cell transplant recipients). Letermovir is a substrate of UGT1A1/3, P-gp, and OATP1B, with its clearance primarily mediated by OATP1B. Letermovir (single-dose) administered with rifampin (single-dose) resulted in increased letermovir exposure through transporter inhibition. Chronic coadministration with rifampin (inhibition plus potential OATP1B induction) resulted in modestly decreased letermovir exposure versus letermovir alone. Letermovir administered 24 hours after last rifampin dose (potential OATP1B induction) resulted in markedly decreased letermovir exposure. These data suggest rifampin may induce transporters that clear letermovir; the modestly reduced letermovir exposure with chronic rifampin coadministration likely reflects the net effect of inhibition and induction. OATP1B endogenous biomarkers coproporphyrin (CP) I and glycochenodeoxycholic acid-sulfate (GCDCA-S) were also analyzed; their exposures increased after single-dose rifampin plus letermovir, consistent with OATP1B inhibition and prior reports of inhibition by rifampin alone. CP I and GCDCA-S exposures were substantially reduced with letermovir administered 24 hours after the last dose of rifampin versus letermovir plus chronic rifampin coadministration, This study suggests that OATP1B induction may contribute to reduced letermovir exposure after chronic rifampin administration, although given the complexity of letermovir disposition, alternative mechanisms are not fully excluded.

Membrane transporters play an important role in the absorption, distribution, metabolism, and excretion of xenobiotic substrates, as well as endogenous compounds. The evaluation of transporter-mediated drug-drug interactions (DDIs) is an important consideration during the drug development process and can guide the safe use of polypharmacy regimens in clinical practice. In recent years, several endogenous substrates of drug transporters have been identified as potential biomarkers for predicting changes in drug transport function and the potential for DDIs associated with drug candidates in early phases of drug development. These biomarker-driven investigations have been applied in both preclinical and clinical studies and proposed as a predictive strategy that can be supplanted in order to conduct prospective DDIs trials. Here we provide an overview of this rapidly emerging field, with particular emphasis on endogenous biomarkers recently proposed for clinically relevant uptake transporters.

Drug transporters play important roles in the elimination of various compounds from the blood. Genetic variation and drug-drug interactions underlie the pharmacokinetic differences for the substrates of drug transporters. Some endogenous substrates of drug transporters have emerged as biomarkers to assess differences in drug transporter activity-not only in animals, but also in humans. Metabolomic analysis is a promising approach for identifying such endogenous substrates through their metabolites. The appropriateness of metabolites is supported by studies in vitro and in vivo, both in animals and through pharmacogenomic or drug-drug interaction studies in humans. This review summarizes current progress in identifying such endogenous biomarkers and applying them to drug transporter phenotyping.

Aim: 2-Hydroxyglutarate (2-HG) is a target engagement biomarker in patients after treatment with inhibitors of mutated isocitrate dehydrogenase (mIDH). Accurate measurement of 2-HG is critical for monitoring the inhibition effectiveness of the inhibitors. Materials & methods: Human plasma samples were spiked with stable isotope labelled internal standard, processed by protein precipitation, and analyzed using LC-MS/MS. This method was validated following regulatory guidance and has been successfully applied in a clinical study for mIDH inhibition. Results: An LC-MS/MS method with a surrogate analyte approach was developed and validated to measure 2-HG in human plasma with acceptable intra- and inter-assay accuracy and precision. Conclusion: A sensitive and robust LC-MS/MS method was developed and validated for measuring 2-HG in human plasma.

To predict the absorption, distribution, metabolism and excretion (ADME) profile of candidate drugs a variety of preclinical models can be applied. The ADME and toxicological behavior of newly developed drugs are often investigated prior to assessment in humans, which is associated with long time-lines and high costs. Therefore, good predictions of ADME profiles earlier in the drug development process are very valuable. Good prediction of intestinal absorption and renal and biliary excretion remain especially difficult, as there is an interplay of active transport and metabolism involved. To study these processes, including enterohepatic circulation, ex vivo tissue models are highly relevant and can be regarded as the bridge between in vitro and in vivo models. In this review the current in vitro, in vivo and in more detail ex vivo models for studying pharmacokinetics in health and disease are discussed. Additionally, we propose novel models, i.e., perfused whole-organs, which we envision will generate valuable pharmacokinetic information in the future due to improved translation to the in vivo situation. These machine-perfused organ models will be particularly interesting in combination with biomarkers for assessing the functionality of transporter and CYP450 proteins.

Metabolomics is a dynamically evolving field, with a major application in identifying biomarkers for drug development and personalized medicine. Numerous metabolomic studies have identified endogenous metabolites that, in principle, are eligible for translation to clinical practice. However, few metabolomic-derived biomarker candidates have been qualified by regulatory bodies for clinical applications. Such interruption in the biomarker qualification process can be largely attributed to various reasons including inappropriate study design and inadequate data to support the clinical utility of the biomarkers. In addition, the lack of robust assays for the routine quantification of candidate biomarkers has been suggested as a potential bottleneck in the biomarker qualification process. In fact, the nature of the endogenous metabolites precludes the application of the current validation guidelines for bioanalytical methods. As a result, there have been individual efforts in modifying existing guidelines and/or developing alternative approaches to facilitate method validation. In this review, three main challenges for method development and validation for endogenous metabolites are discussed, namely matrix effects evaluation, alternative analyte-free matrices, and the choice of internal standards (ISs). Some studies have modified the equations described by the European Medicines Agency for the evaluation of matrix effects. However, alternative strategies were also described; for instance, calibration curves can be generated in solvents and in biological samples and the slopes can be compared through ratios, relative standard deviation, or a modified Stufour suggested approaches while quantifying mainly endogenous metabolitesdent t-test. ISs, on the contrary, are diverse; in which seven different possible types, used in metabolomics-based studies, were identified in the literature. Each type has its advantages and limitations; however, isotope-labeled ISs and ISs created through isotope derivatization show superior performance. Finally, alternative matrices have been described and tested during method development and validation for the quantification of endogenous entities. These alternatives are discussed in detail, highlighting their advantages and shortcomings. The goal of this review is to compare, apprise, and debate current knowledge and practices in order to aid researchers and clinical scientists in developing robust assays needed during the qualification process of candidate metabolite biomarkers. © 2019 John Wiley & Sons Ltd. Mass Spec Rev.