Publications by authors named "Lieve Dillen"

43 Publications

Clinical Investigation on Endogenous Biomarkers to Predict Strong OAT-Mediated Drug-Drug Interactions.

Clin Pharmacokinet 2021 Apr 10. Epub 2021 Apr 10.

Drug Metabolism and Pharmacokinetics, Janssen Pharmaceutical Companies of Johnson & Johnson, Turnhoutseweg 30, 2340, Beerse, Belgium.

Background: Endogenous biomarkers are promising tools to assess transporter-mediated drug-drug interactions early in humans.

Methods: We evaluated on a common and validated in vitro system the selectivity of 4-pyridoxic acid (PDA), homovanillic acid (HVA), glycochenodeoxycholate-3-sulphate (GCDCA-S) and taurine towards different renal transporters, including multidrug resistance-associated protein, and assessed the in vivo biomarker sensitivity towards the strong organic anion transporter (OAT) inhibitor probenecid at 500 mg every 6 h to reach close to complete OAT inhibition.

Results: PDA and HVA were substrates of the OAT1/2/3, OAT4 (PDA only) and multidrug resistance-associated protein 4; GCDCA-S was more selective, having affinity only towards OAT3 and multidrug resistance-associated protein 2. Taurine was not a substrate of any of the investigated transporters under the in vitro conditions tested. Plasma exposure of PDA and HVA significantly increased and the renal clearance of GCDCA-S, PDA and HVA decreased; the magnitude of these changes was comparable to those of known clinical OAT probe substrates. PDA and GCDCA-S were the most promising endogenous biomarkers of the OAT pathway activity: PDA plasma exposure was the most sensitive to probenecid inhibition, and, in contrast, GCDCA-S was the most sensitive OAT biomarker based on renal clearance, with higher selectivity towards the OAT3 transporter.

Conclusions: The current findings illustrate a clear benefit of measuring PDA plasma exposure during phase I studies when a clinical drug candidate is suspected to be an OAT inhibitor based on in vitro data. Subsequently, combined monitoring of PDA and GCDCA-S in both urine and plasma is recommended to tease out the involvement of OAT1/3 in the inhibition interaction.

Clinical Trial Registration: EudraCT number: 2016-003923-49.
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http://dx.doi.org/10.1007/s40262-021-01004-2DOI Listing
April 2021

2020 White Paper on Recent Issues in Bioanalysis: BMV of Hybrid Assays, Acoustic MS, HRMS, Data Integrity, Endogenous Compounds, Microsampling and Microbiome ( - Recommendations on Industry/Regulators Consensus on BMV of Biotherapeutics by LCMS, Advanced Application in Hybrid Assays, Regulatory Challenges in Mass Spec, Innovation in Small Molecules, Peptides and Oligos).

Bioanalysis 2021 Feb 20;13(4):203-238. Epub 2021 Jan 20.

Bristol-Myers Squibb, Pennington, NJ, USA.

The 14 edition of the Workshop on Recent Issues in Bioanalysis (14 WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14 WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by Mass Spectrometry (hybrid assays, LCMS and HRMS) were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication covers the recommendations on (Part 1) Hybrid Assays, Innovation in Small Molecules, & Regulated Bioanalysis. Part 2A (BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation), Part 2B (Regulatory Input) and Part 3 (Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity) are published in volume 13 of Bioanalysis, issues 5, and 6 (2021), respectively.
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http://dx.doi.org/10.4155/bio-2020-0324DOI Listing
February 2021

2-Methyl-pentanoyl-carnitine (2-MPC): a urine biomarker for patent Ascaris lumbricoides infection.

Sci Rep 2020 09 25;10(1):15780. Epub 2020 Sep 25.

Janssen Global Public Health, Janssen R&D, Turnhoutseweg 30, 2340, Beerse, Belgium.

Infections with intestinal worms, such as Ascaris lumbricoides, affect hundreds of millions of people in all tropical and subtropical regions of the world. Through large-scale deworming programs, World Health Organization aims to reduce moderate-to-heavy intensity infections below 1%. Current diagnosis and monitoring of these control programs are solely based on the detection of worm eggs in stool. Here we describe how metabolome analysis was used to identify the A. lumbricoides-specific urine biomarker 2-methyl pentanoyl carnitine (2-MPC). This biomarker was found to be 85.7% accurate in determining infection and 90.5% accurate in determining a moderate-to-heavy infection. Our results also demonstrate that there is a correlation between 2-MPC levels in urine and A. lumbricoides DNA detected in stool. Furthermore, the levels of 2-MPC in urine were shown to rapidly and strongly decrease upon administration of a standard treatment (single oral dose of 400 mg albendazole). In an Ascaris suum infection model in pigs, it was found that, although 2-MPC levels were much lower compared to humans, there was a significant association between urinary 2-MPC levels and both worm counts (p = 0.023) and the number of eggs per gram (epg) counts (p < 0.001). This report demonstrates that urinary 2-MPC can be considered an A. lumbricoides-specific biomarker that can be used to monitor infection intensity.
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http://dx.doi.org/10.1038/s41598-020-72804-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7519643PMC
September 2020

Capillary microsampling in clinical studies: opportunities and challenges in two case studies.

Bioanalysis 2020 Jul 6;12(13):905-918. Epub 2020 Jul 6.

Development Bioanalysis, Janssen Research & Development, A division of Janssen Pharmaceutica NV, Turnhoutseweg 30, Beerse 2340, Belgium.

Capillary microsampling of 15 μl whole blood from fingersticks or heelsticks was used to collect pharmacokinetic (PK) samples from pediatric subjects in two projects. In a mebendazole multisite study in Ethiopia and Rwanda in subjects between 1 and 16 years old, complete PK profiles (7 timepoints) could be obtained, although some of the fingerstick samples were contaminated by the dosing formulation. In a multisite study with a respiratory syncytial virus drug in children between 1 and 24 months old, sparse PK sampling was done (2 samples). All samples were successfully analyzed even though some capillaries were not properly filled. CMS shows potential for PK sampling in pediatrics but may need further optimization.
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http://dx.doi.org/10.4155/bio-2020-0054DOI Listing
July 2020

High-dose etoposide formulations do not saturate intestinal P-glycoprotein: Development, stability, and pharmacokinetics in Sprague-Dawley rats.

Int J Pharm 2020 Jun 4;583:119399. Epub 2020 May 4.

Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark. Electronic address:

It has been suggested that oral absorption of low-permeable P-glycoprotein (P-gp) substrates can be increased through saturation of P-gp. For BCS class IV drug substances, saturating P-gp is challenging due to low aqueous solubility. The present study investigated if the BCS IV drug substance etoposide could be solubilized to a concentration saturating P-gp after oral administration. A formulation consisting of 10% (w/v) of pluronic® F-127 and polyvinylpyrrolidone/vinyl acetate (PVP/VA), and 57% (v/v) ethanol enhanced etoposide's solubility approximately 100 times (16 mg mL) compared to its aqueous solubility. In vitro, this formulation was stable upon dilution in simulated intestinal fluid. In male Sprague-Dawley rats, oral administration of increasing solubilized etoposide doses using the formulation matrix increased the AUC of etoposide dose-proportionally but resulted in a lower absolute oral bioavailability (F) and rate of absorption as compared to control. At the highest investigated dose (100 mg kg), AUC and C were significantly increased by 2.9- and 1.4-fold, respectively, compared to control dosed at 20 mg kg. A single oral dose of 20 mg kg zosuquidar followed by 20 mg kg oral etoposide increased F 8.6-fold. In conclusion, a stable formulation with improved etoposide solubility was developed, yet the formulation did not result in increased oral bioavailability of etoposide.
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http://dx.doi.org/10.1016/j.ijpharm.2020.119399DOI Listing
June 2020

LC-MS quantification of oligonucleotides in biological matrices with SPE or hybridization extraction.

Bioanalysis 2019 Nov;11(21):1941-1954

Development Bioanalysis, Janssen Research & Development, Beerse, Belgium.

Quantitative LC-MS analysis of oligonucleotides (OGNs) in biological matrices is needed to support candidate selection of new therapeutic OGNs. A set of 20 single stranded antisense oligonucleotides (ASO) and five siRNAs were extracted from plasma and tissue homogenates. Anion Exchange (AEX) SPE was selected as generic extraction approach, resulting in recoveries from plasma >70%. Extraction from tissue homogenates showed often more variation and lower recoveries. A proof of concept of a novel tailored hybridization extraction is demonstrated for two 16-mer reference OGNs. Two methods for extraction of OGNs were investigated and applied for quantitative analysis. The AEX-SPE is considered a more generic approach preferred when multiple compounds are evaluated. Hybridization extraction has great potential but critical reagents per analyte are needed.
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http://dx.doi.org/10.4155/bio-2019-0117DOI Listing
November 2019

2019 White Paper on Recent Issues in Bioanalysis: Chromatographic Assays (Part 1 - Innovation in Small Molecules and Oligonucleotides & Mass Spectrometric Method Development Strategies for Large Molecule Bioanalysis).

Bioanalysis 2019 Nov 6;11(22):2029-2048. Epub 2019 Dec 6.

Bristol-Myers Squibb, Princeton, NJ, USA.

The 2019 13 Workshop on Recent Issues in Bioanalysis (WRIB) took place in New Orleans, LA, USA on April 1-5, 2019 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers, immunogenicity and gene therapy. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS, LBA cell-based/flow cytometry assays and qPCR approaches. This 2019 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2019 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations on Innovation in Small Molecules and Oligonucleotides & Mass Spec Method Development Strategies for Large Molecules Bioanalysis. Part 2 (2018 FDA BMV Guidance, 2019 ICH M10 BMV Draft Guideline and regulatory agencies' input on bioanalysis, biomarkers, immunogenicity and gene therapy) and Part 3 (New Insights in Biomarkers Assays Validation, Current & Effective Strategies for Critical Reagent Management, Flow Cytometry Validation in drug discovery & development & CLSI H62, Interpretation of the 2019 FDA Immunogenicity Guidance and The Gene Therapy Bioanalytical Challenges) are published in volume 11 of , issues 23 and 24 (2019), respectively.
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http://dx.doi.org/10.4155/bio-2019-0260DOI Listing
November 2019

Montmorillonite-surfactant hybrid particles for modulating intestinal P-glycoprotein-mediated transport.

Int J Pharm 2019 Nov 13;571:118696. Epub 2019 Sep 13.

Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark. Electronic address:

In the small intestine, P-glycoprotein (P-gp) may limit the permeability of its substrates, which lead to reduced oral absorption. To circumvent the effect of P-gp, a nanocomposite material termed montmorillonite-surfactant hybrid particles was developed. The particles consisted of montmorillonite, the P-gp-inhibiting, nonionic surfactant, polysorbate 20, and the P-gp substrate, digoxin. The present study aimed to investigate if montmorillonite-surfactant hybrid particles could modulate the absorption of digoxin in vivo. Montmorillonite-surfactant hybrid particles were prepared by lyophilising an aqueous suspension of the constituents. Scanning electron microscopy, thermogravimetric analysis, and powder X-ray diffraction revealed an altered surface morphology, decreased water content, and intercalation of polysorbate 20 between montmorillonite layers. The particles were administered orally to Sprague Dawley rats, and digoxin was quantified by liquid chromatography-tandem mass spectrometry. Control digoxin-containing montmorillonite decreased the exposure of digoxin. In contrast, montmorillonite-surfactant hybrid particles increased AUC and C by 31 and 91%, respectively, compared to digoxin in solution. It was hypothesised that montmorillonite-surfactant hybrid particles increased digoxin exposure by forming mucosa-localised elevated concentrations of polysorbate 20 and digoxin, which enhanced the inhibitory effect of polysorbate 20 on P-gp.
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http://dx.doi.org/10.1016/j.ijpharm.2019.118696DOI Listing
November 2019

Comparison of toxicokinetic parameters of a drug and two metabolites following traditional and capillary microsampling in rat.

Bioanalysis 2019 Jun 12;11(13):1233-1242. Epub 2019 Jul 12.

Non-Clinical Safety, Janssen Research & Development, A Division of Janssen Pharmaceutica NV, Turnhoutseweg 30, 2340 Beerse, Belgium.

Following the request of a regulatory authority, a rat study was conducted to compare pharmacokinetic parameters from traditional large volume sampling and capillary microsampling. Rats were dosed with a proprietary compound in three dose groups and blood samples were collected via capillary microsampling (32 μl), immediately followed by traditional large volume sampling (300 μl) up to 24 h postdose. Resulting plasma samples were analyzed for parent drug and two metabolites. AUCs were compared between sampling techniques. There was no statistical difference between AUCs from traditional and microsampling across different doses and analytes.  Toxicokinetic parameters generated from plasma collected as a capillary microsample or traditional large volume sample are highly comparable.
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http://dx.doi.org/10.4155/bio-2019-0085DOI Listing
June 2019

Strategies and analytical workflows to extend the dynamic range in quantitative LC-MS/MS analysis.

Bioanalysis 2019 Jun 17;11(12):1189-1206. Epub 2019 Jun 17.

Janssen Research & Development, 2340 Beerse, Belgium.

To evaluate alternative analytical strategies to extend the dynamic range in quantitative LC-MS/MS. Two approaches based on prior or no prior knowledge of expected exposure levels were evaluated. These approaches make use of two analytical strategies, which include the use of more than one injection volume or dilution of sample extract with solvents or solvent mixtures. A total of 16 compounds with varying logP values were classified into polar and nonpolar groups and used in this evaluation. From the two analytical strategies, three workflows were derived. All three workflows were successfully evaluated and resulted in good accuracy (80-120%) for all the compound groups.
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http://dx.doi.org/10.4155/bio-2018-0309DOI Listing
June 2019

Feedback from the European Bioanalysis Forum liquid microsampling consortium: microsampling: assessing accuracy and precision of handheld pipettes and capillaries.

Bioanalysis 2019 Mar 11;11(6):533-542. Epub 2019 Apr 11.

European Bioanalysis Forum, Havenlaan 86c b204, 1000 Brussels, Belgium.

Microsampling in preclinical pharmacokinetics (PK) studies is currently widely adopted across the pharmaceutical industry. The European Bioanalysis Forum liquid microsampling consortium member companies assessed the accuracy and precision of handheld pipettes and microcapillaries at volumes of less than 10 μl. The following key factors on pipetting performance were also evaluated: Pipette type (positive displacement, air displacement and microcapillary), experience of user and the liquid type. Water was selected as a best-case scenario for accuracy and precision determination and blood plasma as a 'real world' bioanalysis sample type. Accuracy and precision on the pipetted volume decreased at lower volumes and experienced laboratory technicians performed better compared with the infrequent users. With respect to the pipetting devices used, microcapillaries showed better or equivalent accuracy and precision compared with handheld pipettes across the volume range 1-8 μl independent of the matrix used.
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http://dx.doi.org/10.4155/bio-2019-0018DOI Listing
March 2019

Feedback from the European Bioanalysis Forum liquid microsampling consortium: capillary liquid microsampling and assessment of homogeneity of the resultant samples.

Bioanalysis 2019 Mar 11;11(6):525-532. Epub 2019 Apr 11.

European Bioanalysis Forum, Havenlaan 86c b204, 1000 Brussels, Belgium.

Following the completion of a detailed experimental protocol into the potential inhomogeneity of capillary liquid microsamples, which was performed at seven European Bioanalysis Forum member companies, the summary and conclusion on the data are reported here. It has been demonstrated that it is possible to generate homogeneous samples using these microsampling techniques; that the resultant microsamples can be accurate and precise and that capillary liquid microsampling data can be consistent with conventional larger volume plasma samples. However, the data contain some variability which is contributed to by the different range of experiences that each investigating site had with these techniques. Therefore, knowledge of the compounds, well-designed experiments and experience with these techniques are essential for the delivery of high quality data.
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http://dx.doi.org/10.4155/bio-2019-0017DOI Listing
March 2019

2018 White Paper on Recent Issues in Bioanalysis: 'A global bioanalytical community perspective on last decade of incurred samples reanalysis (ISR)' (Part 1 - small molecule regulated bioanalysis, small molecule biomarkers, peptides & oligonucleotide bioanalysis).

Bioanalysis 2018 Nov 29;10(22):1781-1801. Epub 2018 Nov 29.

US FDA, Silver Spring, MD, USA.

The 2018 12 Workshop on Recent Issues in Bioanalysis (12th WRIB) took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day full immersion in bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LC-MS, hybrid ligand binding assay (LBA)/LC-MS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations for LC-MS for small molecules, peptides, oligonucleotides and small molecule biomarkers. Part 2 (hybrid LBA/LC-MS for biotherapeutics and regulatory agencies' inputs) and Part 3 (large molecule bioanalysis, biomarkers and immunogenicity using LBA and cell-based assays) are published in volume 10 of Bioanalysis, issues 23 and 24 (2018), respectively.
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http://dx.doi.org/10.4155/bio-2018-0268DOI Listing
November 2018

Clinical Investigation of Coproporphyrins as Sensitive Biomarkers to Predict Mild to Strong OATP1B-Mediated Drug-Drug Interactions.

Clin Pharmacokinet 2018 12;57(12):1559-1570

Janssen Pharmaceutical Companies of Johnson & Johnson, Turnhoutseweg 30, 2340, Beerse, Belgium.

Introduction: Coproporphyrin (CP) I and III have recently been proposed as endogenous clinical biomarkers to predict organic anion-transporting polypeptide 1B (OATP1B)-mediated drug-drug interactions (DDIs). In the present study, we first investigated the in vitro selectivity of CPI and CPIII towards drug uptake and efflux transporters. We then assessed the in vivo biomarker sensitivity towards OATP1B inhibition.

Methods: To assess transporter selectivity, incubations with CPI and CPIII were performed in vitro, using single transporter-expressing and control systems. Furthermore, CPI and CPIII plasma concentrations were determined from participants of three independent clinical trials who were administered with either a strong, moderate, or mild clinical OATP1B inhibitor.

Results: Our results show that CPI and CPIII are substrates of OATP1B1, OATP1B3, the multidrug resistance-associated protein (MRP) 2, and MRP3. No substrate interaction was shown for other prominent drug transporters that have been associated with clinical DDIs. Results from clinical studies demonstrated that changes in CPI and CPIII plasma levels were predictive for moderate (two to threefold area under the concentration-time curve [AUC] increase) and strong (≥ fivefold increases) clinical OATP1B inhibition. Furthermore, CPI, but not CPIII, concentration changes were predictive for a mild clinically observed DDI where CPI AUC increases of 1.4-fold were comparable with those observed for pitavastatin as victim drug (AUC increases of 1.5-fold).

Conclusion: Our results demonstrate the selectivity of CPI and CPIII towards the OATP1B/MRP pathway, and the herein reported data further underline the potential of CPI and CPIII as selective and sensitive clinical biomarkers to quantify OATP1B-mediated DDIs.
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http://dx.doi.org/10.1007/s40262-018-0648-3DOI Listing
December 2018

Mass spectrometric recommendations for Quan/Qual analysis using liquid-chromatography coupled to quadrupole time-of-flight mass spectrometry.

Anal Chim Acta 2018 Aug 3;1020:62-75. Epub 2018 Mar 3.

Leiden Academic Centre for Drug Research, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.

Background: High-throughput simultaneous quantitative and qualitative (Quan/Qual) analysis is attractive to combine targeted with non-targeted analysis, e.g. in pharmacometabolomics and drug metabolism studies. This study aimed to investigate the possibilities and limitations of high-throughput Quan/Qual analysis by ultra-high performance liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS), to develop a widely applicable Quan/Qual UHPLC-HRMS method and to provide recommendations for Quan/Qual method development.

Methods: A widely applicable 4.25-min UHPLC method for small-molecules was used to investigate and optimize mass spectrometric parameters of a Synapt G2S for Quan/Qual analysis. The method was applied on a rat metabolomics study investigating the effect of the fasting state and administration of a dosing vehicle on the rat plasma metabolic profile.

Results: Highly important parameters for high-throughput Quan/Qual analysis were the scan mode and scan rate. A negative correlation was found between the amount of qualitative information that a method can provide and its quantitative performance (accuracy, precision, sensitivity, linear dynamic range). The optimal balance was obtained using the MS scan mode with a short scan time of 30 ms. This 4.25-min Quan/Qual analysis method enabled quantification with accuracy and precision values ≤ 20% at the lowest quality control (QC) level and ≤15% at higher QC levels for 16 out of 19 tested analytes. It provided both parent m/z values and fragmentation spectra for compound identification with limited loss of chromatographic resolution and it revealed biologically relevant metabolites in its application to the metabolomics study.

Conclusion: Quan/Qual method development requires balancing between the amount of qualitative data, the quality of the quantitative data and the analysis time. Recommendations are provided for MS resolution, scan mode, scan rate, smoothing and peak integration in Quan/Qual method development and analysis.
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http://dx.doi.org/10.1016/j.aca.2018.02.055DOI Listing
August 2018

Development of an LC-MS method to quantify coproporphyrin I and III as endogenous biomarkers for drug transporter-mediated drug-drug interactions.

J Chromatogr B Analyt Technol Biomed Life Sci 2018 Jan 6;1073:80-89. Epub 2017 Dec 6.

Pharmacokinetics, Dynamics & Metabolism, Janssen Research and Development, Division of Janssen Pharmaceutica NV, Turnhoutseweg 30, Beerse, Belgium.

Coproporphyrins are proposed as endogenous biomarkers of hepatic Organic Anion Transporting Polypeptide (OATP)1B functional activity. In this study, a new sample extraction method based on a mixed-mode anion exchange sorbent (SPE clean-up using Oasis 30mg Max 96 well plates) was developed for absolute quantification of coproporphyrin I and III (CP-I and CP-III) in human plasma. Chromatographic separation was performed with an Ace Excel 2 C18 PFP, 3μm, 2.1×150mm, maintained at 60°C. A 10mM ammonium formate containing 0.1% HCOOH and acetonitrile (100%) was used as mobile phase A and B, respectively. Mass transition, m/z 655.3→596.3 was selected to monitor CP-I and CP-III, while m/z 659.3→600.3 transition was used for the stable isotope labelled internal standard. Optimization of the liquid chromatography tandem mass spectrometry method ensured a lower limit of quantification (LLOQ) of 20pg/mL. Both CP-I and CP-III had extraction recoveries of 70%. The calibration range was 0.02-100ng/mL for both CP-I and CP-III, yielding calibration curves with correlation coefficients greater than 0.988. Inter day precision (CV<9%) and accuracy (84.3-103.9%) complied with the recommendation of the European Bioanalytical Forum. The optimized method was used to analyse plasma samples originating from three independent clinical studies. Obtained CP-I and CP-III plasma baseline levels in healthy volunteers were in good agreement with previously published data. Moreover, CP-I and CP-III plasma levels in human subjects dosed with a clinically confirmed OATP inhibitor were significantly increased compared to their baseline levels. These data demonstrate the potential of CP-I and CP-III as endogenous biomarkers to predict the drug-drug interaction (DDI) related to hepatic OATP1B inhibition. Stability of CP-I and CP-III in plasma and solvents under different processing and storage conditions was also evaluated.
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http://dx.doi.org/10.1016/j.jchromb.2017.12.008DOI Listing
January 2018

Quantitative analysis of imetelstat in plasma with LC-MS/MS using solid-phase or hybridization extraction.

Bioanalysis 2017 Dec 5;9(23):1859-1872. Epub 2017 Dec 5.

Development Bioanalysis, Janssen Research & Development, 2340 Beerse, Belgium.

Aim: Imetelstat, a 13-mer oligonucleotide with a lipid tail is being evaluated for treating hematologic myeloid malignancies. This report describes the development of extraction and quantification methods for imetelstat. Methodology & results: Imetelstat was extracted using SPE (rat plasma) or by hybridization using a biotinylated capture probe (human plasma) and was quantified by LC-MS/MS. Calibration curves were established (0.1-50 μg/ml). Stability of imetelstat in plasma was demonstrated. Concentrations of imetelstat extracted using either of the methods and quantified with LC-MS/MS were comparable with a validated ELISA.

Conclusion: Two extraction methods (solid phase and hybridization) were developed for quantifying imetelstat in plasma using LC-MS/MS. The hybridization extraction in combination with LC-MS/MS is a novel extraction approach.
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http://dx.doi.org/10.4155/bio-2017-0145DOI Listing
December 2017

High sensitivity and selectivity in quantitative analysis of drugs in biological samples using 4-column multidimensional micro-UHPLC-MS enabling enhanced sample loading capacity.

Anal Chim Acta 2017 Oct 4;989:104-111. Epub 2017 Aug 4.

Janssen Research & Development, Turnhoutseweg 30, 2340 Beerse, Belgium.

Sensitivity is often a critical parameter in quantitative bioanalyses in drug development. For liquid-chromatography-based methods, sensitivity can be improved by reducing the column diameter, but practical sensitivity gains are limited by the reduced sample loading capacity on small internal diameter (I.D.) columns. We developed a set-up that has overcome these limitations in sample loading capacity. The set-up uses 4 columns with gradually decreasing column diameters along the flow-path (2.1 → 1 → 0.5 → 0.15 mm). Samples are pre-concentrated on-line on a 2.1 mm I.D. trapping column and back flushed to a 1 mm I.D. UHPLC analytical column and separated. The peak(s) of interest are transferred using heartcutting to a second trapping column (0.5 mm I.D.), which is back-flushed to a 0.15 mm I.D. micro-UHPLC analytical column for orthogonal separation. The proof of concept of the set-up was demonstrated by the simultaneous analysis of midazolam and 1'-hydroxy midazolam in plasma by injection of 80 μL of protein precipitated plasma. The 4-column funnel set-up proved to be robust and resulted in a 10-50 times better sensitivity compared to a trap-elute approach and 250-500 fold better compared to direct micro-UHPLC analysis. A lower limit of quantification of 100 fg/mL in plasma was obtained for both probe compounds.
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http://dx.doi.org/10.1016/j.aca.2017.07.067DOI Listing
October 2017

Outsourcing bioanalytical services at Janssen Research and Development: the sequel anno 2017.

Bioanalysis 2017 Aug 1;9(15):1195-1201. Epub 2017 Aug 1.

Development Bioanalysis, Janssen R&D, Turnhoutseweg 30, 2340 Beerse, Belgium.

The strategy of outsourcing bioanalytical services at Janssen has been evolving over the last years and an update will be given on the recent changes in our processes. In 2016, all internal GLP-related activities were phased out and this decision lead to the re-orientation of the in-house bioanalytical activities. As a consequence, in-depth experience with the validated bioanalytical assays for new drug candidates is currently gained together with the external partner, since development and validation of the assay and execution of GLP preclinical studies are now transferred to the CRO. The evolution to externalize more bioanalytical support has created opportunities to build even stronger partnerships with the CROs and to refocus internal resources. Case studies are presented illustrating challenges encountered during method development and validation at preferred partners when limited internal experience is obtained or with introduction of new technology.
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http://dx.doi.org/10.4155/bio-2017-0082DOI Listing
August 2017

The application of capillary microsampling in GLP toxicology studies.

Bioanalysis 2017 Apr 16;9(7):531-540. Epub 2017 Mar 16.

Development Bioanalysis, Janssen Research & Development, Division of Janssen Pharmaceutica NV, Turnhoutseweg 30, 2340 Beerse, Belgium.

Aim: Capillary microsampling (CMS) to collect microplasma volumes is gradually replacing traditional, larger volume sampling from rats in GLP toxicology studies.

Methodology: About 32 µl of blood is collected with a capillary, processed to plasma and stored in a 10- or 4-µl capillary which is washed out further downstream in the laboratory. CMS has been standardized with respect to materials, assay validation experiments and application for sample analysis.

Conclusion: The implementation of CMS has resulted in blood volume reductions in the rat from 300 to 32 µl per time point and the elimination of toxicokinetic satellite groups in the majority of the rat GLP toxicology studies. The technique has been successfully applied in 26 GLP studies for 12 different projects thus far.
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http://dx.doi.org/10.4155/bio-2016-0297DOI Listing
April 2017

Assessment of light stability of drugs in blood and plasma.

Bioanalysis 2016 Oct 9;8(19):2007-21. Epub 2016 Sep 9.

Janssen Research & Development, Turnhoutseweg 30, 2340 Beerse, Belgium.

Aim: A procedure was developed for the assessment of photochemical stability of drugs in blood and plasma under standardized conditions. The procedure avoids a variable outcome of photochemical stability experiments and tests relevant worst case conditions so that unnecessary light protection is avoided. Results/methodology: Blood and plasma were spiked with a mixture of drugs and incubated in a Suntest CPS(+), in the laboratory on the bench and near the window on a sunny summer day. The results were compared.

Discussion/conclusion: No protection from light, limited protection from light and full protection from light are advised for drugs that are stable in plasma in the Suntest CPS(+) at 250 W/m(2) for at least 30 min, for 5-30 min and for <5 min, respectively.
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http://dx.doi.org/10.4155/bio-2016-0109DOI Listing
October 2016

Evaluation of the diagnostic potential of urinary N-Acetyltyramine-O,β-glucuronide (NATOG) as diagnostic biomarker for Onchocerca volvulus infection.

Parasit Vectors 2016 05 23;9(1):302. Epub 2016 May 23.

Janssen Diagnostics, Janssen R&D, Turnhoutseweg 30, 2340, Beerse, Belgium.

Background: Onchocerciasis, also known as river blindness is one of the neglected tropical diseases affecting millions of people, mainly in sub-Saharan Africa and is caused by the filarial nematode Onchocerca volvulus. Efforts to eliminate this disease are ongoing and are based on mass drug administration programs with the microfilaricide ivermectin. In order to monitor the efficacy of these programs, there is an unmet need for diagnostic tools capable of identifying infected patients. We have investigated the diagnostic potential of urinary N-acetyltyramine-O,β-glucuronide (NATOG), which is a promising O. volvulus specific biomarker previously identified by urine metabolome analysis.

Methods: A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was used to assess the stability characteristics of NATOG and to evaluate the levels of NATOG in study samples. An LC-fluorescence method was also developed.

Results: Stability characteristics of NATOG were investigated and shown to be ideally suited for use in tropical settings. Also, an easy and more accessible method based on liquid chromatography coupled to fluorescence detection was developed and shown to have the necessary sensitivity (limit of quantification 1 μM). Furthermore, we have evaluated the levels of NATOG in a population of 98 nodule-positive individuals from Ghana with no or low levels of microfilaria in the skin and compared them with the levels observed in different control groups (endemic controls (n = 50), non-endemic controls (n = 18) and lymphatic filariasis (n = 51). Only a few (5 %) of nodule-positive individuals showed an increased level (> 10 μM) of NATOG and there was no statistical difference between the nodule-positive individuals and the control groups (P > 0.05).

Conclusions: Results of the present study indicate the limited potential of NATOG as a diagnostic biomarker for O. volvulus infection in amicrofilaridermic individuals.
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http://dx.doi.org/10.1186/s13071-016-1582-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4877973PMC
May 2016

Quantitation of Bedaquiline: Points of Attention.

Clin Infect Dis 2016 07 29;63(1):145-6. Epub 2016 Mar 29.

Janssen Research and Development, Department of Bioanalysis, Turnhoutseweg 30, 2340 Beerse, Belgium.

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http://dx.doi.org/10.1093/cid/ciw164DOI Listing
July 2016

Half-life extension of the HIV-fusion inhibitor peptide TRI-1144 using a novel linker technology.

Eur J Pharm Biopharm 2015 Jun 18;93:254-9. Epub 2015 Apr 18.

ProLynx LLC, 455 Mission Bay Blvd. South, Suite 145, San Francisco, CA 94158, USA. Electronic address:

We have previously developed a linker technology for half-life extension of peptides, proteins and small molecule drugs (1). The linkers undergo β-elimination reactions with predictable cleavage rates to release the native drug. Here we utilize this technology for half-life extension of the 38 amino acid HIV-1 fusion inhibitor TRI-1144. Conjugation of TRI-1144 to 40 kDa PEG by an appropriate β-eliminative linker and i.v. administration of the conjugate increased the in vivo half-life of the released peptide from 4 to 34 h in the rat, and the pharmacokinetic parameters were in excellent accord with a one-compartment model. From these data we simulated the pharmacokinetics of the PEG-TRI-1144 conjugate in humans, predicting a t1/2,β of 70 h for the released peptide, and that a serum concentration of 25 nM could be maintained by weekly doses of 8 μmol of the conjugate. Using a non-circulating carrier (2) similar simulations indicated a t1/2,β of 150 h for the peptide released from the conjugate and that dosing of only 1.8 μmol/week could maintain serum concentrations of TRI-1144 above 25 nM. Hence, releasable β-eliminative linkers provide significant half-life extension to TRI-1144 and would be expected to do likewise for related peptides.
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http://dx.doi.org/10.1016/j.ejpb.2015.04.003DOI Listing
June 2015

Systematic evaluation of commercially available ultra-high performance liquid chromatography columns for drug metabolite profiling: optimization of chromatographic peak capacity.

J Chromatogr A 2014 Dec 20;1374:122-133. Epub 2014 Nov 20.

Leiden Academic Centre for Drug Research, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands; Pharmacokinetics, Dynamics and Metabolism, Janssen R&D, Turnhoutseweg 30, 2340 Beerse, Belgium.

The present study investigated the practical use of modern ultra-high performance liquid chromatography (UHPLC) separation techniques for drug metabolite profiling, aiming to develop a widely applicable, high-throughput, easy-to-use chromatographic method, with a high chromatographic resolution to accommodate simultaneous qualitative and quantitative analysis of small-molecule drugs and metabolites in biological matrices. To this end, first the UHPLC system volume and variance were evaluated. Then, a mixture of 17 drugs and various metabolites (molecular mass of 151-749Da, logP of -1.04 to 6.7), was injected on six sub-2μm particle columns. Five newest generation core shell technology columns were compared and tested against one column packed with porous particles. Two aqueous (pH 2.7 and 6.8) and two organic mobile phases were evaluated, first with the same flow and temperature and subsequently at each column's individual limit of temperature and pressure. The results demonstrated that pre-column dead volume had negligible influence on the peak capacity and shape. In contrast, a decrease in post-column volume of 57% resulted in a substantial (47%) increase in median peak capacity and significantly improved peak shape. When the various combinations of stationary and mobile phases were used at the same flow rate (0.5mL/min) and temperature (45°C), limited differences were observed between the median peak capacities, with a maximum of 26%. At higher flow though (up to 0.9mL/min), a maximum difference of almost 40% in median peak capacity was found between columns. The finally selected combination of solid-core particle column and mobile phase composition was chosen for its selectivity, peak capacity, wide applicability and peak shape. The developed method was applied to rat hepatocyte samples incubated with the drug buspirone and demonstrated to provide a similar chromatographic resolution, but a 6 times higher signal-to-noise ratio than a more traditional UHPLC metabolite profiling method using a fully porous particle packed column, within one third of the analysis time. In conclusion, a widely applicable, selective and fast chromatographic method was developed that can be applied to perform drug metabolite profiling in the timeframe of a quantitative analysis. It is envisioned that this method will in future be used for simultaneous qualitative and quantitative analysis and can therefore be considered a first important step in the Quan/Qual workflow.
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http://dx.doi.org/10.1016/j.chroma.2014.11.037DOI Listing
December 2014

EBF: reflection on bioanalytical assay requirements used to support liquid microsampling.

Bioanalysis 2014 ;6(19):2581-6

Bioanalytical Science & Toxicokinetics, Drug Metabolism & Pharmacokinetics, GlaxoSmithKline Research & Development, Ware, UK.

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http://dx.doi.org/10.4155/bio.14.211DOI Listing
July 2015

European Bioanalysis Forum continued plans to support liquid microsampling.

Bioanalysis 2014 ;6(14):1897-900

Janssen Research & Development, Turnhoutseweg, 30, B2340 Beerse, Belgium.

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http://dx.doi.org/10.4155/bio.14.99DOI Listing
May 2015

Bioanalysis for plasma protein binding studies in drug discovery and drug development: views and recommendations of the European Bioanalysis Forum.

Bioanalysis 2014 Mar;6(5):673-82

TNO Triskelion BV, Utrechtseweg 48, 3704 HE Zeist, The Netherlands.

Plasma protein binding (PPB) is an important parameter for a drug's efficacy and safety that needs to be investigated during each drug-development program. Even though regulatory guidance exists to study the extent of PPB before initiating clinical studies, there are no detailed instructions on how to perform and validate such studies. To explore how PPB studies involving bioanalysis are currently executed in the industry, the European Bioanalysis Forum (EBF) has conducted three surveys among their member companies: PPB studies in drug discovery (Part I); in vitro PPB studies in drug development (Part II); and in vivo PPB studies in drug development. This paper reflects the outcome of the three surveys, which, together with the team discussions, formed the basis of the EBF recommendation. The EBF recommends a tiered approach to the design of PPB studies and the bioanalysis of PPB samples: 'PPB screening' experiments in (early) drug discovery versus qualified/validated procedures in drug development.
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http://dx.doi.org/10.4155/bio.13.338DOI Listing
March 2014

Blood microsampling using capillaries for drug-exposure determination in early preclinical studies: a beneficial strategy to reduce blood sample volumes.

Bioanalysis 2014 Feb;6(3):293-306

Drug Metabolism & Pharmacokinetics, Janssen Research & Development, Janssen Pharmaceutica N.V., Turnhoutseweg 30, 2340 Beerse, Belgium.

Background: Capillary microsampling (CMS) of blood with subsequent blood analysis offers a potential strategy to deal with increased demand to reduce blood sample volumes in animal discovery and preclinical studies.

Results: A generic approach is presented allowing PK analysis in 15 µl blood samples. CMS blood exposure data were compared with the traditional plasma exposure results in rats and dogs. Blood PK profiles obtained for two different compounds were in agreement with profiles obtained in plasma. From these studies ex vivo blood to plasma ratios were also obtained. In a mouse study, blood PK profiles that were obtained following automatic sampling overlay with the blood PK profiles obtained with CMS.

Conclusion: CMS in 15 µl glass capillaries allows collection and handling of small and exact volumes of blood. Although CMS can also be applied for plasma collection, the full benefit is only achieved with blood collection and analysis.
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http://dx.doi.org/10.4155/bio.13.286DOI Listing
February 2014