Pharmacokinetics of JNJ-73763989 and JNJ-56136379 (Bersacapavir) in Participants With Moderate Hepatic Impairment.

JNJ-73763989 is comprised of 2 short interfering RNAs (siRNAs), JNJ-73763976 and JNJ-73763924, that target hepatitis B virus (HBV) mRNAs for degradation, thereby inhibiting HBV replication. JNJ-56136379 is a capsid assembly modulator that inhibits HBV replication by inducing the formation of empty capsids (CAM-E). In 2 phase 1, open-label, non-randomized, single-center studies, the single-dose pharmacokinetics, safety, and tolerability of JNJ-73763989 or JNJ-56136379 were assessed in participants with moderate hepatic impairment (Child-Pugh Class B) versus participants with normal liver function. Participants in both studies received a single subcutaneous dose of JNJ-73763989 200 mg or oral JNJ-56136379 250 mg, followed by an evaluation of plasma pharmacokinetic parameters and safety assessments. Plasma exposure to JNJ-73763976, JNJ-73763924, and JNJ-56136379 was 1.3- to 1.4-, 1.8- to 2.2-, and 1.1- to 1.3-fold higher in participants with moderate hepatic impairment versus participants with normal liver function; however, these increases were not considered clinically relevant. Both drugs were well tolerated and safe, with 7 (21.9%) participants experiencing 1 or more treatment-emergent adverse events, 3 of which were related to JNJ-56136379. Overall, the plasma exposures of JNJ-73763989 and JNJ-56136379 were higher in participants with moderate hepatic impairment, but both were well tolerated. Further studies are needed to evaluate the effect of hepatic impairment under multiple-dose administration.

Pharmacokinetics, Safety, and Tolerability of the siRNA JNJ-73763989 in Healthy Chinese Adult Participants.

JNJ-73763989, composed of the 2 short-interfering RNA triggers JNJ-73763976 and JNJ-73763924, targets all hepatitis B virus messenger RNAs, thereby reducing all viral proteins. In this phase 1, single-site, open-label, parallel-group, randomized study, participants were given 1 subcutaneous injection of JNJ-73763989 (100 or 200 mg) to investigate the pharmacokinetics, safety, and tolerability of JNJ-73763989 in healthy Chinese adult participants. Plasma and urine pharmacokinetic parameters were determined for each trigger up to 48 hours after dosing. Eighteen participants, 9 per dose group, were enrolled. The median age and weight were 33.0 years and 73.65 kg; 83.3% were male. Exposure of both triggers increased dose proportionally. Median time to maximum concentration ranged from 6.0 to 10.0 hours, and mean elimination half-life ranged from 4.5 to 4.8 hours across both triggers and doses. Mean urinary excretion for JNJ-73763976 and JNJ-73763924 ranged from 17.7% to 19.4% and 13.1% to 13.2% for the 100- and 200-mg dose groups, respectively. All treatment-emergent adverse events (AEs) were mild and resolved by study end, and no AEs or serious AEs resulted in premature study discontinuation or death. Overall, the pharmacokinetics of JNJ-73763989 in healthy Chinese participants were consistent with previous studies, and JNJ-73763989 was generally safe and well tolerated after a single dose.

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

Aim: Quantitative LC-MS analysis of oligonucleotides (OGNs) in biological matrices is needed to support candidate selection of new therapeutic OGNs. Methodology & results: 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. Conclusion: 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.

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

Aim: To evaluate alternative analytical strategies to extend the dynamic range in quantitative LC-MS/MS. Methods & results: 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. Conclusion: All three workflows were successfully evaluated and resulted in good accuracy (80-120%) for all the compound groups.

Mycotoxin contamination of sorghum and its contribution to human dietary exposure in four sub-Saharan countries.

This research aimed at evaluating the safety, and the type, level and prevalence of mycotoxins in grain sorghum of four sub-Saharan African (SSA) countries (Burkina Faso, Ethiopia, Mali and Sudan). A multi-analyte LC-MS/MS method for quantification of 23 mycotoxins (nivalenol, deoxynivalenol, fusarenon X, neosolaniol, 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, diacetoxyscirpenol, roquefortine C, HT-2 toxin, alternariol, T-2 toxin, FB1, FB2, FB3, zearalenone, aflatoxin G1, aflatoxin G2, aflatoxin B1, aflatoxin B2, sterigmatocystin, OTA, altenuene, alternariol monomethylether) was applied to different sorghum matrices. Of the 1533 analysed samples, 33% were contaminated with at least one of the following mycotoxins: aflatoxins, fumonisins, sterigmatocystin, Alternaria toxins, OTA and zearalenone. Country of origin, colour, source and collection period of sorghum samples significantly influenced the type, level and prevalence of mycotoxins. Sterigmatocystin (15%), fumonisins (17%) and aflatoxins (13%) were the most prevalent. FB1 (274 ± 585 µg/kg) had the highest mean concentration followed by FB2 (214 ± 308 µg/kg) while diacetoxyscirpenol (8.12 ± 19.2 µg/kg) and HT-2 (11.9 ± 0.00 µg/kg) had the lowest concentrations. Neosolaniol, fusarenon-X, 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, T-2 toxin, nivalenol and roquefortine C were not detected in any of the samples. Sudan had the lowest prevalence and mean concentration of all mycotoxins. Pink sorghum had the highest concentrations of fumonisins and aflatoxins. Mycotoxins from Aspergillus spp. and Alternaria spp. are the mycotoxins of concern in SSA grain sorghum with regard to prevalence, concentration and possible health risk from exposure. Based on the performed risk characterisation, daily consumption of sorghum containing aflatoxins, alternariol, alternariol monomethyl ether, sterigmatocystin and OTA could result in exceeding the established health-based guidance values for these toxins.

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

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.

Ultra-High-Performance Supercritical Fluid Chromatography as a Separation Tool for Fusarium Mycotoxins and Their Modified Forms.

A simple, reliable method for the detection of free and modified Fusarium mycotoxins in beer using state-of-the-art ultra-high-performance supercritical fluid chromatography (UHPSFC) with low-resolution tandem MS (MS/MS) is presented in this paper. The UHPSFC-MS/MS method was developed for nivalenol, deoxynivalenol, 15-acetyl-deoxynivalenol, 3-acetyl-deoxynivalenol, deoxynivalenol-3-glucoside, HT-2 toxin, T-2 toxin, T-2 toxin-3-glucoside, neosolaniol, diacetoxyscirpenol, zearalenone, α-zearalenol, and ß-zearalenol and their internal standards deepoxy-deoxynivalenol and zearalanone. Due to the broad range of the physicochemical properties of the aforementioned, the sample preparation step was minimized to avoid analyte losses. Extraction with acetonitrile-water-acetic acid (79 + 20 + 1, v/v/v) and hexane in combination with solid-phase extraction (C18) was followed by a filtration step. After filtration, the extract was evaporated, and the remaining residue was redissolved in a mobile phase for injection (methanol-water; 90 + 10, v/v). A mobile phase consisting of supercritical CO2 and a small portion of methanol was used. The developed multimycotoxin method permits the simultaneous determination of multiple fusariotoxins in an one-step chromatographic run using UHPSFC-MS/MS. SFC is a promising strategy; however, the retention mechanism is complex, leading to the unpredictable nature of elution and to some mycotoxins not being retained on the column. This restricts the applicability of UHPSFC in multimycotoxin analyses. The present study is the first report on the use of UHPSFC for the analysis of free and modified Fusarium mycotoxins.

Dissipation kinetics and degradation mechanism of amicarbazone in soil revealed by a reliable LC-MS/MS method.

A sensitive and reliable analytical method was developed for simultaneous determination of amicarbazone (AMZ) and its two major metabolites including desamino amicarbazone (DA) and isopropyl-2-hydroxy-DA-amicarbazone (Ipr-2-OH-DA-AMZ) in soil for the first time. Targeted analytes were extracted and purified using a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) procedure, and then analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a total run time of 9 min. The established approach was extensively validated by determining the linearity (R (2) ≥ 0.99), recovery (84-96 ), sensitivity (limits of quantification at 5-10 µg kg(-1)), and precision (RSDs ≤12 %). Based on the methodological advances, the subsequent dissipation kinetics and degradation mechanism of amicarbazone in soil were thoroughly investigated in an illumination incubator. As revealed, AMZ was easily degraded with the half-lives of 13.9-19.7 days in soil. Field trial results of AMZ (40 g a.i. ha(-1)) in Shanghai showed that the residues of AMZ and its metabolite Ipr-2-OH-DA-AMZ decreased from 0.505 mg kg(-1) (day 50) to 0.038 mg kg(-1) (day 365) and from 0.099 mg kg(-1) (day 50) to 0.028 mg kg(-1) (day 365), respectively, while the content of DA increased from 0.097 mg kg(-1) (day 50) to 0.245 mg kg(-1) (day 365). This study provided valuable data to understand the toxicity of AMZ and substantially promote its safe application to protect environment and human health.

Cumulative health risk assessment of co-occurring mycotoxins of deoxynivalenol and its acetyl derivatives in wheat and maize: case study, Shanghai, China.

Humans are naturally and frequently exposed to a multitude of mycotoxins, but health risk assessments are usually performed on individual mycotoxins, which may underestimate the total risks. In this study, we assessed for the first time the cumulative health risks of concomitant exposure via dietary intake (DI) to multiple mycotoxins, namely deoxynivalenol (DON) and its acetyl derivatives of 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON), based on the concentration addition (CA) concept. A cross-sectional study was conducted in seven districts in Shanghai, China with 1269 participants and 330 wheat and maize samples analyzed. After probabilistic analysis using Monte Carlo simulation, the results showed no health risks to the population in Shanghai considering individual mycotoxins. However, if the cumulative health risks were calculated based on the combined consideration of DON with either 3-ADON or 15-ADON or both, the DI values in 95th percentile were up to 1087 ng/kg body weight/day, exceeding the Provisional Maximum Tolerable Daily Intake (PMTDI) of 1000 ng/kg body weight/day and hence representing potential health risks to the population in Shanghai. The integrated study proposed here could be a model strategy for cumulative health risk assessment on the co-occurring hazards in the fields of food safety combined with environmental contaminants.

Development and validation of an ultra-high-performance liquid chromatography tandem mass spectrometric method for the simultaneous determination of free and conjugated Alternaria toxins in cereal-based foodstuffs.

A UPLC-ESI+/--MS/MS method for the simultaneous determination of free (alternariol, alternariol monomethyl ether, altenuene, tenuazonic acid, tentoxin, altertoxin-I) and conjugated (sulfates and glucosides of alternariol and alternariol monomethyl ether) Alternaria toxins in cereals and cereal products (rice, oat flakes and barley) was developed. Optimization of the sample preparation and extraction methodology was achieved through experimental design, using full factorial design for extraction solvent composition optimization and fractional factorial design to identify the critical factors in the sample preparation protocol, which were in turn subjected to optimization. Final extracts were analysed using an Waters Acquity UPLC system coupled to a Quattro Premier XE mass spectrometer equipped with an electrospray interface operated in both positive and negative ionization mode. Chromatographic separation was achieved using an Acquity UPLC HSS T3 column, and the applied gradient elution programme allowed for the simultaneous determination of 10 Alternaria toxins in a one-step chromatographic run with a total run time of only 7min. Subsequently, the method, applying isotopically labelled internal standards ([2H4]-alternariol monomethyl ether and [13C6,15N]-tenuazonic acid), was validated for several parameters such as linearity, apparent recovery, limit of detection, limit of quantification, precision, measurement uncertainty and specificity (in agreement with the criteria mentioned in Commission Regulation No. 401/2006/EC and Commission Decision No. 2002/657/EC). During validation, quality of the bioanalytical data was improved by counteracting the observed heteroscedasticity through the application of weighted least squares linear regression (WLSLR). Finally, 24 commercially available cereal-based foodstuffs were subjected to analysis, revealing the presence of tenuazonic acid in both rice and oat flake samples (

A comprehensive study to explore differences in mycotoxin patterns from agro-ecological regions through maize, peanut, and cassava products: a case study, Cameroon.

A total of 420 samples were collected from agrarian households. Whereas 51% (215/420) of the samples were contaminated with one or more toxins, the contamination rates for maize, peanut, and cassava products were 74, 62, and 24%, respectively. The fumonisins (20-5412 µg/kg), aflatoxin B1 (6-645 µg/kg), roquefortine C (1-181 µg/kg), and deoxynivalenol (27-3842 µg/kg) were the most prevalent contaminants in maize. For peanut samples, aflatoxin B1 (6-125 µg/kg) and ochratoxin A (0.3-12 µg/kg) were the main contaminants, whereas aflatoxin B1 (6-194 µg/kg) and penicillic acid (25-184 µg/kg) were detected in the cassava products. Exposures calculated through maize intake for fumonisin B1 and aflatoxin B1 were several-fold higher (2-5 for fumonisin B1 and 10(4)-10(5) for aflatoxin B1) than the health-based guidance values of 2 µg/kg bw/day and 0.15 ng/kg bw/day, respectively. The study design constitutes a good model that can be implemented in other sub-Saharan African countries.

Development and application of salting-out assisted liquid/liquid extraction for multi-mycotoxin biomarkers analysis in pig urine with high performance liquid chromatography/tandem mass spectrometry.

Direct determination of urinary mycotoxins is a better approach to assess individual's exposure than the indirect estimation from average dietary intakes. In this study, a new analytical method was developed and validated for simultaneous analysis of aflatoxin B1, deoxynivalenol, fumonisin B1, ochratoxin A, zearalenone and T2 toxin and their metabolites in pig urine. In total 12 analytes were selected. A salting-out assisted liquid-liquid extraction procedure was used for sample preparation. High performance liquid chromatography/tandem mass spectrometry was used for the separation and detection of all the analytes. The extraction recoveries were in a range of 70-108%, with the intra-day relative standard deviation and inter-day relative standard deviation lower than 25% for most of the compounds at 3 different concentration levels. Meanwhile the method bias for all the analytes did not exceed 20%. The limits of quantification ranged from 0.07ngmL(-1) for ochratoxin A to 3.3ngmL(-1) for deoxynivalenol. Matrix effect was evaluated in this study and matrix-matched calibration was used for quantification. The developed method was also validated for human urine as an extension of its application. Finally, the developed method was applied in a pilot study to analyze 28 pig urine samples. Deoxynivalenol, aflatoxin B1, fumonisin B1 and ochratoxin A were detected in these samples.

A direct assessment of mycotoxin biomarkers in human urine samples by liquid chromatography tandem mass spectrometry.

Detection of mycotoxin biomarkers in urine of humans and animals provides a direct approach for assessing exposure to these mycotoxins as opposed to the indirect approach of food analysis, which in most cases is affected by the heterogeneity of the toxin in the food samples. Seven (7) mycotoxins and their metabolites (total 18 analytes) were selected and an LC-MS/MS method for their determination in human urine was developed and validated. The method consisted of direct analysis of two mycotoxin conjugates, deoxynivalenol-glucuronide and zearalenone-glucuronide without beta glucuronidase digestion of the urine samples. Since high method sensitivity is of utmost importance in such study, critical factors which could improve the analyte recovery and method sensitivity were investigated by a D-optimal experimental design. Urine samples (10 mL) were first extracted with 15 mL ethyl acetate/formic acid (99/1, v/v) followed by SAX SPE clean-up of the acidified aqueous fraction. Both extracts were combined and analyzed using an LC-MS/MS system operated in the positive ionization mode. A total run time of 28 min was adopted with all the 18 analytes eluting within 15 min. The method was validated by taking into consideration the guidelines specified in Commission Decision 2002/657/EC and 401/2006/EC. Forty samples obtained from volunteers within the laboratory research group were analyzed as part of a pilot study. All results were expressed per mg creatinine. A total of 9 samples were found contaminated with one or more of the following analytes: DON, OTA, OTα, 4-OH OTA, ZEN, CIT and ß-ZOL. One-eighth (5/40) of the samples were contaminated with DON in the range of 3.7-67 ng mg(-1) creatinine. Samples with detectable levels of DON did not show any co-occurrence of DON-3Glu. One sample was found to be contaminated with 4-OH OTA (

A validated multianalyte LC-MS/MS method for quantification of 25 mycotoxins in cassava flour, peanut cake and maize samples.

This study was designed to develop a sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous detection and quantification of 25 mycotoxins in cassava flour, peanut cake and maize samples with particular focus on the optimization of the sample preparation protocol and method validation. All 25 mycotoxins were extracted in a single step with a mixture of methanol/ethyl acetate/water (70:20:10, v/v/v). The method limits of quantification (LOQ) varied from 0.3 µg/kg to 106 µg/kg. Good precision and linearity were observed for most of the mycotoxins. The method was applied for the analysis of naturally contaminated peanut cake, cassava flour and maize samples from the Republic of Benin. All samples analyzed (fifteen peanut cakes, four maize flour and four cassava flour samples) tested positive for one or more mycotoxins. Aflatoxins (total aflatoxins; 10-346 µg/kg) and ochratoxin A (