Therapeutic siRNA Loaded to RISC as Single and Double Strands Requires an Appropriate Quantitative Assay for RISC PK Assessment.

In recent years, therapeutic siRNA projects are booming in the biotech and pharmaceutical industries. As these drugs act by silencing the target gene expression, a critical step is the binding of antisense strands of siRNA to RNA-induced silencing complex (RISC) and then degrading their target mRNA. However, data that we recently obtained suggest that double-stranded siRNA can also load to RISC. This brings a new understanding of the mechanism of RISC loading which may have a potential impact on how quantification of RISC loaded siRNA should be performed. By combining RNA immune precipitation and probe-based hybridization LC-fluorescence approach, we have developed a novel assay that can accurately quantify the RISC-bound antisense strand, irrespective of which form (double-stranded or single-stranded) is loaded on RISC. In addition, this novel assay can discriminate between the 5'-phosphorylated antisense (5'p-AS) and the nonphosphorylated forms, therefore specifically quantifying the RISC bound 5'p-AS. In comparison, stem-loop qPCR assay does not provide discrimination and accurate quantification when the oligonucleotide analyte exists as a mixture of double and single-stranded forms. Taking together, RISC loading assay with probe-hybridization LC-fluorescence technique would be a more accurate and specific quantitative approach for RISC-associated pharmacokinetic assessment.

JNJ-73763989 pharmacokinetics and safety: Liver-targeted siRNAs against hepatitis B virus, in Japanese and non-Japanese healthy adults, and combined with JNJ-56136379 and a nucleos(t)ide analogue in patients with chronic hepatitis B.

BACKGROUND: JNJ-73763989 comprises two hepatitis B virus (HBV)-specific, liver-targeted N-galactosamine-conjugated short interfering RNA triggers, JNJ-73763976 and JNJ-73763924. JNJ-73763989 pharmacokinetics, safety and tolerability were assessed in two phase 1 studies: Japanese (NCT04002752), and non-Japanese healthy participants and chronic hepatitis B (CHB) patients also receiving the HBV capsid assembly modulator JNJ-56136379 and a nucleos(t)ide analogue (NA) (NCT03365947). METHODS: Healthy participant cohorts were double-blind and randomized to receive a single subcutaneous JNJ-73763989 dose (non-Japanese participants, 35, 100, 200, 300 or 400 mg; Japanese participants, 25, 100 or 200 mg) or placebo. JNJ-73763976 and JNJ-73763924 plasma concentrations were assessed over 48 h. CHB patients received JNJ-73763989 200 mg every 4 weeks plus daily oral JNJ-56136379 250 mg and NA in an open-label fashion. Safety and tolerability were assessed through Day 28 (healthy participants) or Day 112 (patients). RESULTS: Thirty non-Japanese (n = 4/dose; placebo, n = 10) and 24 Japanese healthy participants (n = 6/dose; placebo, n = 6) were randomized. JNJ-73763976 and JNJ-73763924 exposure generally increased in a dose-proportional manner. Mean plasma half-life was 4-9 h. No differences between pharmacokinetic parameters were apparent between non-Japanese and Japanese healthy participants. In the 12 CHB patients, mean JNJ-73763976, JNJ-73763924 and JNJ-56136379 plasma concentrations 2 h post-dose on Day 29 were 663, 269 and 14,718 ng/mL, respectively. In both studies, all adverse events were mild/moderate. CONCLUSION: JNJ-73763976 and JNJ-73763924 had short plasma half-lives and exposure generally increased in a dose-proportional manner; there were no pharmacokinetic differences between Japanese and non-Japanese healthy adults. JNJ-73763989 with or without JNJ-56136379 and NA was generally safe and well tolerated.

Multimodal biomarker discovery for active Onchocerca volvulus infection.

The neglected tropical disease onchocerciasis, or river blindness, is caused by infection with the filarial nematode Onchocerca volvulus. Current estimates indicate that 17 million people are infected worldwide, the majority of them living in Africa. Today there are no non-invasive tests available that can detect ongoing infection, and that can be used for effective monitoring of elimination programs. In addition, to enable pharmacodynamic studies with novel macrofilaricide drug candidates, surrogate endpoints and efficacy biomarkers are needed but are non-existent. We describe the use of a multimodal untargeted mass spectrometry-based approach (metabolomics and lipidomics) to identify onchocerciasis-associated metabolites in urine and plasma, and of specific lipid features in plasma of infected individuals (O. volvulus infected cases: 68 individuals with palpable nodules; lymphatic filariasis cases: 8 individuals; non-endemic controls: 20 individuals). This work resulted in the identification of elevated concentrations of the plasma metabolites inosine and hypoxanthine as biomarkers for filarial infection, and of the urine metabolite cis-cinnamoylglycine (CCG) as biomarker for O. volvulus. During the targeted validation study, metabolite-specific cutoffs were determined (inosine: 34.2 ng/ml; hypoxanthine: 1380 ng/ml; CCG: 29.7 ng/ml) and sensitivity and specificity profiles were established. Subsequent evaluation of these biomarkers in a non-endemic population from a different geographical region invalidated the urine metabolite CCG as biomarker for O. volvulus. The plasma metabolites inosine and hypoxanthine were confirmed as biomarkers for filarial infection. With the availability of targeted LC-MS procedures, the full potential of these 2 biomarkers in macrofilaricide clinical trials, MDA efficacy surveys, and epidemiological transmission studies can be investigated.

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

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.

2-methyl butyramide, a previously identified urine biomarker for Ascaris lumbricoides, is not present in infected Indonesian individuals.

ᅟ: Previous reports suggest that the 2-methyl butyramide and 2-methyl valeramide metabolites of Ascaris lumbricoides in urine of infected individuals could be considered as urinary biomarkers for active infection. We have developed an LC-MS method with a detection limit of 10 ng/mL using synthetic chemicals as reference material. Urine samples (n = 21) of infected individuals were analyzed for the presence of these metabolites, but they were not detected in any of the samples. Furthermore, the recorded 1H-NMR spectrum for reference 2-methyl butyramide did not match with the spectrum that was described for the Ascaris metabolite. Based on these two observations, we concluded that the urinary biomarkers that were detected for A. lumbricoides infection are not 2-methyl butyramide nor 2-methylvaleramide. New discovery efforts will be required to identify the structure of these metabolite biomarkers in urine of infected individuals. TRIAL REGISTRATION: Urine samples used in this study were collected as part of a clinical trial with trial number ISRCTN75636394 (12 November 2013).

Sensitive Flow-through Immunoassay for Rapid Multiplex Determination of Cereal-borne Mycotoxins in Feed and Feed Ingredients.

An easy-to-operate membrane-based flow-through test for multiplex screening of four mycotoxins (zearalenone, deoxynivalenol, aflatoxin B1, and ochratoxin A) in a variety of cereal-based feed ingredients and compound feeds, such as wheat, barley, soybean, wheat bran, rice, rice bran, maize, rapeseed meal, and sunflower meal, and various types of complete feed (duckling feed, swine feed, broiler feed, piglet feed) was developed and validated. First, the antibodies were evaluated by enzyme-linked immunosorbent assay and then employed in the membrane rapid test. The cutoff levels for zearalenone, deoxynivalenol, aflatoxin B1, and ochratoxin A were 50, 200, 1, and 10 µg/kg, respectively, based on European regulations and consumers' requirements. As sample pretreatment, consecutive steps of extraction, dilution, solid-phase extraction by addition of C18 sorbent, and final filtration of supernatant were followed. Both the sample preparation and the analysis procedure were simple, cost-effective, and easy to perform on-site in a nonlaboratory environment. The impact of sample processing on the result of the experiment was investigated supported by experimental design. The validation procedure was performed on the basis of Commission Regulation 2006/401/EC. The numbers of false-positive and false-negative outcomes were <5%, going along with the Commission Decision 2002/657/EC. Liquid chromatography-tandem mass spectrometry was performed as a confirmatory technique.

Keeping mycotoxins away from the food: Does the existence of regulations have any impact in Africa?

Following the discovery of aflatoxins in the early 1960s, there have been many studies leading to the uncovering of many mycotoxins and the understanding of associated health effects in animals and humans. Consequently, there has been a global increase in the number of countries with mycotoxin regulations in foods. However, many African countries have only regulations for aflatoxins (or a few other mycotoxins) in specific foods, or no regulations at all. This paper critically reviews the challenges thwarting the establishment of mycotoxin regulations and their impacts on human dietary mycotoxin exposure in Africa. Mycotoxin regulatory limits for different countries are compared with mycotoxin tolerable daily intakes established by international food safety bodies taking into account consumption patterns. The agrarian setup, food insecurity, and mycotoxin analytical challenges in African countries are discussed; and more feasible mycotoxin dietary exposure reduction strategies are proposed.

Validated UPLC-MS/MS Methods To Quantitate Free and Conjugated Alternaria Toxins in Commercially Available Tomato Products and Fruit and Vegetable Juices in Belgium.

Ultraperformance liquid chromatography tandem mass spectrometry and Quick, Easy, Cheap, Effective, Rugged, and Safe based analytical methodologies to quantitate both free (alternariol (1), alternariol monomethyl ether (2), tenuazonic acid (3), tentoxin (4), altenuene (5), altertoxin-I (6)) and conjugated (sulfates and glucosides of 1 and 2) Alternaria toxins in fruit and vegetable juices and tomato products were developed and validated. Acceptable limits of quantitation (0.7-5.7 µg/kg), repeatability (RSDr < 15.7%), reproducibility (RSDR < 17.9%), and apparent recovery (87.0-110.6%) were obtained for all analytes in all matrices investigated. 129 commercial foodstuffs were analyzed, and 3 was detected in 100% of tomato product samples (

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

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,ß-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.

Toxigenic potentiality of Aspergillus flavus and Aspergillus parasiticus strains isolated from black pepper assessed by an LC-MS/MS based multi-mycotoxin method.

A liquid chromatography triple quadrupole tandem mass spectrometry method was developed and validated to determine mycotoxins, produced by fungal isolates grown on malt extract agar (MEA). All twenty metabolites produced by different fungal species were extracted using acetonitrile/1% formic acid. The developed method was applied to assess the toxigenic potentiality of Aspergillus flavus (n = 11) and Aspergillus parasiticus (n = 6) strains isolated from black peppers (Piper nigrum L.) following their growth at 22, 30 and 37 °C. Highest mean radial colony growth rates were observed at 30 °C for A. flavus (5.21 ± 0.68 mm/day) and A. parasiticus (4.97 ± 0.33 mm/day). All of the A. flavus isolates produced aflatoxin B1 and O-methyl sterigmatocystin (OMST) while 91% produced aflatoxin B2 (AFB2) and 82% of them produced sterigmatocystin (STERIG) at 30 °C. Except one, all the A. parasiticus isolates produced all the four aflatoxins, STERIG and OMST at 30 °C. Remarkably high AFB1 was produced by some A. flavus isolates at 22 °C (max 16-40 mg/kg). Production of mycotoxins followed a different trend than that of growth rate of both species. Notable correlations were found between different secondary metabolites of both species; R(2) 0.87 between AFB1 and AFB2 production. Occurrence of OMST could be used as a predictor for AFB1 production.

Effectiveness of hand sorting, flotation/washing, dehulling and combinations thereof on the decontamination of mycotoxin-contaminated white maize.

Maize is one of the major staple foods of Sub-Saharan Africa and is consumed as whole or dehulled grain. In this region, where the environmental conditions favour fungal growth and mycotoxin production, the majority of the population are subsistence consumers who, unfortunately, have little or no access to mycotoxin testing of their food. In an attempt to develop feasible reduction strategies in dietary mycotoxin exposure of the population, a three-factorial design experiment was conducted to examine and compare the efficacy of hand sorting, flotation, dehulling and combinations thereof in removing naturally occurring aflatoxins, fumonisins, nivalenol, deoxynivalenol and alternariol in shelled white maize. Regression analysis was used to determine the significant (p < 0.05) process variables on the removal of mycotoxins from the maize. Results from this experiment indicated that hand sorting had the greatest effect on mycotoxin removal, while flotation yielded the least effect. In particular hand sorting left < 6% of aflatoxin B1 and < 5% of fumonisin B1. Based on these results, hand sorting of maize grains is being recommended as a last line of defence against mycotoxin exposure among subsistence consumers.

A multi-analyte LC-MS/MS method for the analysis of 23 mycotoxins in different sorghum varieties: the forgotten sample matrix.

An LC-MS/MS method was developed and validated for the detection and quantification of 23 mycotoxins in different varieties of sorghum. The method performance characteristics were as follows: suitable linearity ranges for all 23 mycotoxins with p-value >0.05; limits of detection (1.2-50 µg/kg), limits of quantification (2.5-100 µg/kg), repeatability (RSDr, 7-22%), intermediate precision (RSDR, 14-44%) and apparent recovery (0.2-11%, expressed as bias). The method was applied to analyze 10 samples obtained from retail shops in Belgium (n=8) and Germany (n=2). Nine of the 10 samples (90%) were positive for the following mycotoxins: aflatoxin B1 (50 µg/kg), alternariol monomethyl ether (

Uncommon occurrence ratios of aflatoxin B1, B 2, G 1, and G 2 in maize and groundnuts from Malawi.

We report an unusual aflatoxin profile in maize and groundnuts from Malawi, with aflatoxin G1 found routinely at equal or even higher levels than aflatoxin B1. Aflatoxin B1 (AFB1) ratio in a contaminated sample is generally greater than 50% of total aflatoxin (sum of aflatoxin B1, B2, G1, and G2). In Malawi, the aflatoxin occurrence ratios were determined by examining LC-MS/MS and HPLC fluorescence detection (FLD) data of 156 naturally contaminated raw maize and 80 groundnut samples collected in 2011 and 2012. Results showed that natural aflatoxin occurrence ratio differed. In 47% of the samples, the concentration of AFG1 was higher than that of AFB1. The mean concentration percentages of AFB1/AFB2/AFG1/AFG2 in reference to total aflatoxins were found to be 47:5:43:5%, respectively. The AFG1 and AFB1 50/50 trend was observed in maize and groundnuts and was consistent for samples collected in both years. If the AFB1 measurement was used to check compliance of total aflatoxin regulatory limit set at 10, 20, 100, and 200 µg/kg with an assumption that AFB1≥50% of the total aflatoxin content, 8, 13, 24, and 26% false negative rates would have occurred respectively. It is therefore important for legislation to consider total aflatoxins rather than AFB1 alone.

Multiplex lateral flow immunoassay for mycotoxin determination.

A new lateral flow immunoassay (LFA) is proposed for qualitative and/or semiquantitative determination of aflatoxin B1 (AFB1), zearalenone (ZEA), deoxynivalenol (DON), and their analogues (AFs, ZEAs, DONs) in cereal samples. Each of the mycotoxin specific antibody was class specific and there was no cross reactivity to other groups of compounds. The visual limits of detection (vLOD) of the strip were 0.03, 1.6, and 10 µg/kg for AFB1, ZEA and DON, respectively. The calculated limits of detection (cLOD) were 0.05, 1, and 3 µg/kg, respectively. Meanwhile the cutoff values were achieved at 1, 50, and 60 µg/kg for AFB1, ZEA and DON, respectively. Recoveries ranged from 80% to 122% and RSD from 5% to 20%. Both the vLOD and cLOD for the three mycotoxins were lower than the EU maximum levels. Analysis of naturally contaminated maize samples resulted in a good agreement between the multiplex LFA and LC-MS/MS (100% for DONs and AFs, and 81% for ZEAs). Careful analysis of the results further explained the general overestimation of LFA compared to chromatographic methods for quantification of mycotoxins.

Multimycotoxin analysis in urines to assess infant exposure: a case study in Cameroon.

This study was conducted to investigate mycotoxin exposure in children (n=220, aged 1.5-4.5years) from high mycotoxin contamination regions of Cameroon and to examine the association between the mycotoxin levels (in total 18 analytes) and several socio-demographic factors and anthropometric characteristics. A cross-sectional study was conducted in six villages in Cameroon with 220 children. Mycotoxins and their metabolites were detected in 160/220 (73%) urine samples. There were significant differences in the mean contamination levels of ochratoxin A (p=0.01) and ß-zearalenol (p=0.017) between the two agro-ecological zones investigated. Likewise significant differences were observed in the mean levels of aflatoxin M1 (p=0.001) across the weaning categories of these children. The mean concentration of aflatoxin M1 detected in the urine of the partially breastfed children (1.43ng/mL) was significantly higher (p=0.001) than those of the fully weaned children (0.282ng/mL). Meanwhile, the mean concentrations of deoxynivalenol (3.0ng/mL) and fumonisin B1 (0.59ng/mL) detected in the urine of the male children was significantly (p value 0.021 for deoxynivalenol and 0.004 for fumonisin B1) different from the levels detected in the urine of female children; 0.71ng/mL and 0.01ng/mL for deoxynivalenol and fumonisin B1 respectively. In this study, there was no association between the different malnutrition categories (stunted, wasting and underweight) and the mycotoxin concentrations detected in the urine of these children. However, there is sufficient evidence to suggest that children in Cameroon under the age 5 are exposed to high levels of carcinogenic substances such as fumonisin B1, aflatoxin M1 and ochratoxin A through breastfeeding. To the best of our knowledge, this is the first report of its kind carried out in West Africa to determine multi-mycotoxin exposure in infants.