Single Multifunctional Nanocabinets-Based Target-Activated Feedback for Simultaneously Precise Monitoring and Therapy.

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.

STRATEGIES AND CHALLENGES IN METHOD DEVELOPMENT AND VALIDATION FOR THE ABSOLUTE QUANTIFICATION OF ENDOGENOUS BIOMARKER METABOLITES USING LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY.

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.

Endogenous Biomarkers for SLC Transporter-Mediated Drug-Drug Interaction Evaluation.

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.

Towards human ex vivo organ perfusion models to elucidate drug pharmacokinetics in health and disease.

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.

Validation of a Gravitational Model to Study Local Endogenous Biomarkers in Chronic Venous Insufficiency.

OBJECTIVE/BACKGROUND: Unlike most systemic chronic diseases, chronic venous insufficiency (CVI) is ideal to study using endogenous biomarkers. The stimulus causing damage can be turned on and off with gravitational positioning and venous blood samples can be taken locally. Annexin V (apoptosis) and microparticles (cell membrane debris) were used as markers of cell destruction, with matrix metalloproteinases (MMPs) as markers of tissue remodelling. The aim of this proof of concept study was to validate a gravitational model by investigating whether standing induced biochemical stress and whether recovery occurs on lying and after compression. METHODS: Fourteen patients (C4a-b) and 14 volunteers (C0-1) were tested under three supervised laboratory conditions for 1 h on separate days: (i) stationary standing on a small paper square; (ii) lying with both legs elevated 20°; (iii) compression standing using a 23-32 mmHg below knee stocking. Immediately after each condition, venous blood was withdrawn from the ankle. Commercial enzyme linked immunosorbent assay kits were used for batch analysis of the plasma samples. RESULTS: Median (interquartile range [IQR]) values of annexin V (AU/mL) and microparticles (nM) standing were as follows: volunteers 2.9 (2 - 3.4) and 10.2 (8.8 - 13.8), and patients 2.2 (1.3 - 6) and 11.3 (7.7 - 20), respectively. Significant reductions were observed lying: volunteers 2.1 (1.5 - 2.7; p = .019) and 8.5 (7.4 - 9.4; p = .041), patients 1.7 (1.2 - 2.7; p = .004) and 8.5 (7.0 - 11.4; p = .041), respectively. Globally, all median MMP values in the patients reduced with lying and with compression versus standing (p = .004). Individually, significant reductions occurred in MMPs 2 and 13 with compression and MMPs 3, 7, 8, 9, 10, and 12 on lying. Lying was more effective at reducing MMP levels than compression. CONCLUSION: Annexin V and microparticle concentrations are responsive to elevation and compression after 1 h. In the patients, all the tested MMPs decreased after lying and with compression versus standing. This model provides evidence supporting gravitational protection in the treatment of CVI.

Investigation of the protective effect of Paeonia lactiflora on Semen Strychni-induced neurotoxicity based on monitoring nine potential neurotoxicity biomarkers in rat serum and brain tissue.

Semen Strychni has been widely used as a traditional Chinese herb medicine, but its clinical use was limited for its potential neurotoxicity and nephrotoxicity. This study aimed to investigate S. Strychni-induced neurotoxicity and the neuro-protective effect of Paeonia lactiflora based on monitoring nine potential neurotoxicity biomarkers in rat serum and brain tissue. A sensitive liquid chromatography-tandem mass spectrometry method was developed and validated to monitor serotonin, tryptophan, dopamine, tyrosine and glutamate in serum and five brain regions (prefrontal cortex, hippocampus, striatum, cerebellum and hypothalamus). Analytes were separated on a CAPCELL CORE PC column (150 mm × 2 mm, 2.7 µm) with a gradient program of acetonitrile-water (0.2 % formic acid) and a total runtime of 7.5 min. In addition, enzyme-linked immunosorbent assay was conducted to determine four kinds of protein (tryptophan hydroxylase, tyrosine hydroxylase, endogenous brain-derived neurotrophic factor and nerve growth factor). Results demonstrated that the administration of S. Strychni could cause certain endogenous substances disorder. These analytes were found significantly changed (p < 0.05) in serum (except glutamate) and in certain tested brain regions in S. Strychni extract group. Pretreatment of P. lactiflora could significantly reverse the S. Strychni-induced neurotoxicity and normalize the levels of such endogenous substances. The study could be further used in predicting and monitoring neurotoxicity caused by other reasons, and it was expected to be useful for improving clinical use of S. Strychni through pretreatment with P. lactiflora.

Expanding Role of Endogenous Biomarkers for Assessment of Transporter Activity in Drug Development: Current Applications and Future Horizon.

The evaluation of transporter-mediated drug-drug interactions (DDIs) during drug development and post-approval contributes to benefit-risk assessment and helps formulate clinical management strategies. The use of endogenous biomarkers, which are substrates of clinically relevant uptake and efflux transporters, to assess the transporter inhibitory potential of a drug has received widespread attention. Endogenous biomarkers, such as coproporphyrin (CP) I and III, have increased mechanistic understanding of complex DDIs. Other endogenous biomarkers are under evaluation, including, but not limited to, sulfated bile acids and 4-pyridoxic acid (PDA). The role of endogenous biomarkers has expanded beyond facilitating assessment of transporter-mediated DDIs and they have also been used to understand alterations in transporter activity in the setting of organ dysfunction and various disease states. We envision that endogenous biomarker-informed approaches will not only help to formulate a prudent and informed DDI assessment strategy but also facilitate quantitative predictions of changes in drug exposures in specific populations.

Innovative Approaches to Optimize Clinical Transporter Drug-Drug Interaction Studies.

Of the 450 cell membrane transporters responsible for shuttling substrates, nutrients, hormones, neurotransmitters, antioxidants, and signaling molecules, approximately nine are associated with clinically relevant drug-drug interactions (DDIs) due to their role in drug and metabolite transport. Therefore, a clinical study evaluating potential transporter DDIs is recommended if an investigational product is intestinally absorbed, undergoes renal or hepatic elimination, or is suspected to either be a transporter substrate or perpetrator. However, many of the transporter substrates and inhibitors administered during a DDI study also affect cytochrome P450 (CYP) activity, which can complicate data interpretation. To overcome these challenges, the assessment of endogenous biomarkers can help elucidate the mechanism of complex DDIs when multiple transporters or CYPs may be involved. This perspective article will highlight how creative study designs are currently being utilized to address complex transporter DDIs and the role of physiology-based -pharmacokinetic (PBPK) models can play.

A Metabolomics Approach for Predicting OATP1B-Type Transporter-Mediated Drug-Drug Interaction Liabilities.

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.

Determination of the endogenous OATP1B biomarkers glycochenodeoxycholate-3-sulfate and chenodeoxycholate-24-glucuronide in human and mouse plasma by a validated UHPLC-MS/MS method.

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.

Using Endogenous Biomarkers to Derisk Assessment of Transporter-Mediated Drug-Drug Interactions: A Scientific Perspective.

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.

Current progress in identifying endogenous biomarker candidates for drug transporter phenotyping and their potential application to drug development.

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.

Quantitation of 2-hydroxyglutarate in human plasma via LC-MS/MS using a surrogate analyte approach.

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.

Biomarker Discovery for Cytochrome P450 1A2 Activity Assessment in Rats, Based on Metabolomics.

Cytochrome P450 1A2 (CYP1A2) is one of the major CYP450 enzymes (CYPs) in the liver, and participates in the biotransformation of various xenobiotics and endogenous signaling molecules. The expression and activity of CYP1A2 show large individual differences, due to genetic and environmental factors. In order to discover non-invasive serum biomarkers associated with hepatic CYP1A2, mass spectrometry-based, untargeted metabolomics were first conducted, in order to dissect the metabolic differences in the serum and liver between control rats and ß-naphthoflavone (an inducer of CYP1A2)-treated rats. Real-time reverse transcription polymerase chain reaction and pharmacokinetic analysis of phenacetin and paracetamol were performed, in order to determine the changes of mRNA levels and activity of CYP1A2 in these two groups, respectively. Branched-chain amino acids phenylalanine and tyrosine were ultimately focalized, as they were detected in both the serum and liver with the same trends. These findings were further confirmed by absolute quantification via a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based targeted metabolomics approach. Furthermore, the ratio of phenylalanine to tyrosine concentration was also found to be highly correlated with CYP1A2 activity and gene expression. This study demonstrates that metabolomics can be a potentially useful tool for biomarker discovery associated with CYPs. Our findings contribute to explaining interindividual variations in CYP1A2-mediated drug metabolism.

Mass isotopomer-guided decluttering of metabolomic data to visualize endogenous biomarkers of drug toxicity.

Metabolomics offers the opportunity to uncover endogenous biomarkers that can lead to metabolic pathways and networks and that underpin drug toxicity mechanisms. A novel protocol is presented and discussed that is applicable to drugs which generate urinary metabolites when administered to mice sensitive to its toxicity. The protocol would not apply to drugs that are not metabolized or eliminated by a different route. Separate stable isotope-labeled and unlabeled drug administration to mice is made together with collection of urines from control animals. Untargeted mass spectrometry-based metabolomic analysis of these three urine groups is conducted in addition to principal components analysis (PCA). In the case of unlabeled acetaminophen and [acetyl-2H3]acetaminophen, each given at a hepatotoxic dose (400 mg/kg i.p.) to the sensitive mouse strain (wild-type 129), the PCA loadings plot showed a distribution of ions in the shape of a "fallen-Y" with the deuterated metabolites in one arm and the paired nondeuterated metabolites in the other arm of the fallen-Y. Ions corresponding to the endogenous toxicity biomarkers sat in the mouth of the fallen-Y. This protocol represents an innovative means to separate endogenous biomarkers from drug metabolites, thereby aiding the identification of biomarkers of drug toxicity. For acetaminophen, increased hepatic oxidative stress, mitochondrial damage, Ca2+ signaling, heme catabolism, and saturation of glucuronidation, together with decreased fatty acid ß-oxidation and cellular energy dysregulation were all implied from the discovered biomarkers. The protocol can be applied to other drugs and may now be translated to clinical studies.

LC-MS/MS assay for N1-methylnicotinamide in humans, an endogenous probe for renal transporters.

BACKGROUND: N1-methylnicotinamide (1-NMN) has been proposed as a potential clinical biomarker to assess drug-drug interactions involving organic cation transporters (OCT2) and multidrug and toxin extrusion protein transporters. RESULTS: A hydrophilic interaction liquid chromatography-MS/MS assay, to quantify 1-NMN, in human plasma and urine is reported. MATERIALS & METHODS: A hydrophilic interaction chromatography (HILIC)-tandem mass spectrometry (MS/MS) assay to quantify 1-NMN in human plasma and urine is reported. The basal 1-NMN levels in plasma and urine were 4-120 and 2000-15,000 ng/ml, respectively. CONCLUSION: 1-NMN plasma AUCs increased two- to fourfold versus placebo following the administration of a clinical candidate that in vitro experiments indicated was an OCT2 inhibitor. The described hydrophilic interaction liquid chromatography-MS/MS assay can be used to assess a clinical compound candidate for the inhibition of OCT2 and multidrug and toxin extrusion protein transporter in first-in-human studies.

Identification of Endogenous Biomarkers to Predict the Propensity of Drug Candidates to Cause Hepatic or Renal Transporter-Mediated Drug-Drug Interactions.

Drug transporters expressed in liver and kidney play a critical role in the elimination of a wide range of drugs and xenobiotics and inhibition of these transporters may therefore cause clinically significant drug-drug interactions (DDIs). Currently, in vitro transporter inhibition data are used to assess the risk that a drug candidate may act as an inhibitor of a transporter in patients at clinically relevant exposures. However, this approach is hampered by low confidence in in vitro to in vivo extrapolations, and large inter-system and inter-laboratory variability in in vitro data. Several endogenous compounds have been identified as substrates of drug transporters. Determining the impact of perpetrator drugs on the plasma or urinary exposure of these potential endogenous biomarkers in humans is being explored as an alternative approach to assess the DDI liability of drug candidates, especially in early drug development. In this review, we provide an overview of recently identified biomarkers used to study the inhibition of hepatic and renal transporters; summarize the methods and strategies employed to identify biomarkers; and discuss the utility, limitation, and future direction of biomarker approaches to predict transporter-mediated DDIs.

Endogenous Biomarkers to Assess Drug-Drug Interactions by Drug Transporters and Enzymes.

BACKGROUND: Drug-Drug Interactions (DDI) by modulation of drug transporters or drug metabolizing enzymes are common in multi-drug therapy. DDI potential of any new drug is assessed by conducting separate clinical studies using relevant probe substrates, which involves additional resource and cost. Recently, several endogenous compounds have been evaluated as substrates of transporters and enzymes that could be assessed as part of early clinical trials along with the assessment of drug pharmacokinetics, pharmacodynamics and safety studies. This enables an early readout on potential DDIs avoiding or minimally delaying the conduct of definitive DDI studies until later in clinical development. METHOD: This review describes various endogenous biomarkers reported for drug transporters and metabolizing enzymes with their advantages and limitations. CONCLUSION: Furthermore, the authors describe strategies to adopt while exploring a new endogenous biomarker, and factors to be considered in selection of biomarkers with the current challenges and opportunities.