Metabolic Soft Spot and Pharmacokinetics: Functionalization of C-3 Position of an Eph–Ephrin Antagonist Featuring a Bile Acid Core as an Effective Strategy to Obtain Oral Bioavailability in Mice

UniPR129, an L-β-homotryptophan conjugate of the secondary bile acid lithocholic acid (LCA), acts as an effective protein-protein interaction (PPI) inhibitor of the Eph–ephrin system but suffers from a poor oral bioavailability in mice. To improve UniPR129 bioavailability, a metabolic soft spot, i.e., the 3α-hydroxyl group on the LCA steroidal ring, was functionalized to 3-hydroxyimine. In vitro metabolism of UniPR129 and 3-hydroxyimine derivative UniPR500 was compared in mouse liver subcellular fractions, and main metabolites were profiled by high resolution (HR-MS) and tandem (MS/MS) mass spectrometry. In mouse liver microsomes (MLM), UniPR129 was converted into several metabolites: M1 derived from the oxidation of the 3-hydroxy group to 3-oxo, M2–M7, mono-hydroxylated metabolites, M8–M10, di-hydroxylated metabolites, and M11, a mono-hydroxylated metabolite of M1. Phase II reactions were only minor routes of in vitro biotransformation. UniPR500 shared several metabolic pathways with parent UniPR129, but it showed higher stability in MLM, with a half-life (t1/2) of 60.4 min, if compared to a t1/2 = 16.8 min for UniPR129. When orally administered to mice at the same dose, UniPR500 showed an increased systemic exposure, maintaining an in vitro valuable pharmacological profile as an EphA2 receptor antagonist and an overall improvement in its physico-chemical profile (solubility, lipophilicity), if compared to UniPR129. The present work highlights an effective strategy for the pharmacokinetic optimization of aminoacid conjugates of bile acids as small molecule Eph–ephrin antagonists.

The contamination rate of Aflatoxin M1 in milk samples of Iran: A systematic review and meta-analysis protocol

Aflatoxin M1 (AFM1) as a 4-hydroxylated metabolite of carcinogenic mycotoxin Aflatoxin B1 (AFB1) is considered as an acute toxic compound found in milk and its derived products (1). Regarding the critical public health issue induced by AFM1 and as milk is essential in human food chain, its maximum limit has been set by regulatory agencies considering the economic status and development of the countries (4). The maximum permissible limit for AFM1 is 50 ng/kg (different countries including European Union (EU) members) to 100 ng/kg (different countries including USA) (5). In Iran, it has been regulated as 100 ng/kg (6). To the best of our knowledge, there is several similar systematic review and meta-analysis about the contamination of campylobacter in milk, globally and specifically in Iran (4, 5, 7-10). However, we will try to examine the mean and prevalence of AFM1 in different type of milk (cow, sheep, goat, raw, processed) in different region of Iran with long time interval (1974-2021). Moreover, more general and specific databases and grey literature with excellent eligibility criteria will be applied to have a more comprehensive review leading to an interesting finding by techniques such as risk of bias assessment (for assessing quality of included studies), publication bias assessment, finding the source of heterogeneity by sub-group analysis in addition to meta-analysis process.

Assessment of aflatoxin exposure using urine biomarker in pregnant and non-pregnant women in Yazd, Center of Iran

Background: Aflatoxins (AFs) are one of the most prevalent toxins, which long-term exposure to them could be a risk factor for liver cancer. AFM1 is the hydroxylated metabolite of AFB1 , therefore, the presence of AFM1 in urine samples can give an appropriate estimation of dietary AF exposure in human. Methods: The present study aimed to evaluate the excretion level of AFM1 in urine samples of pregnant and non-pregnant women in Yazd, Iran. A total of 85 urine samples (42 pregnant and 43 non-pregnant) were selected randomly from women who had referred to health centers of Yazd during March to May 2017. From each participant, a 72-hour dietary recall was asked and the data were recorded and later analyzed by ELISA kits. Results: The results showed that the mean level of AFM1 in pregnant and non-pregnant women was 8.23±2.9 and 35.5±1.05 pg mL-1, respectively. Excretion of AFM1 in urine samples had a significant relationship with some demographic factors and type of consumed foods (P<0.05). Conclusion: There was a significant relationship between the education level, place of residence, and the consumption of nuts with the excretion of AFM1 . It can be concluded that some foods distributed in Yazd are contaminated with AFs, and a significant number of people are exposed to high concentrations of AFM1 .

Phase I In Vitro Metabolic Profiling of the Synthetic Cannabinoid Receptor Agonists CUMYL-THPINACA and ADAMANTYL-THPINACA

Synthetic cannabinoid receptor agonists (SCRAs) remain popular drugs of abuse. As many SCRAs are known to be mostly metabolized, in vitro phase I metabolic profiling was conducted of the two indazole-3-carboxamide SCRAs: CUMYL-THPINACA and ADAMANTYL-THPINACA. Both compounds were incubated using pooled human liver microsomes. The sample clean-up consisted of solid phase extraction, followed by analysis using liquid chromatography coupled to a high resolution mass spectrometer. In silico-assisted metabolite identification and structure elucidation with the data-mining software Compound Discoverer was applied. Overall, 28 metabolites were detected for CUMYL-THPINACA and 13 metabolites for ADAMATYL-THPINACA. Various mono-, di-, and tri-hydroxylated metabolites were detected. For each SCRA, an abundant and characteristic di-hydroxylated metabolite was identified as a possible in vivo biomarker for screening methods. Metabolizing cytochrome P450 isoenzymes were investigated via incubation of relevant recombinant liver enzymes. The involvement of mainly CYP3A4 and CYP3A5 in the metabolism of both substances were noted, and for CUMYL-THPINACA the additional involvement (to a lesser extent) of CYP2C8, CYP2C9, and CYP2C19 was observed. The results suggest that ADAMANTYL-THPINACA might be more prone to metabolic drug−drug interactions than CUMYL-THPINACA, when co-administrated with strong CYP3A4 inhibitors.

Determination of Aflatoxins by Liquid Chromatography Coupled to High-Resolution Mass Spectrometry

The most common mycotoxins are aflatoxins (AFs), which are produced by strains of various species of molds in the genus Aspergillus (A. flavus, A. parasiticus, A. nomius and A. tamarii) and can grow on many foods, mainly peanuts, maize and cottonseed. AFs are currently considered to be the most hazardous mycotoxins to health, in particular because of their hepatocellular carcinogenic potential. The main aflatoxins are B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) although many other derivatives have been described. In addition, animals consuming contaminated feeds are able to metabolize them by hydroxylation in a certain position, yield for example aflatoxin M1 (AFM1) and aflatoxin M2 (AFM2) from AFB1 and AFB2, respectively. Nowadays, only the four main AFs and one hydroxylated metabolite (AFM1) are routinely analyzed. High resolution mass spectrometry (HRMS) using Orbitrap or time-of-flight (TOF) mass analysers is a trend for AFs determination, allowing to determine AFs and their derivatives for which there are no commercial standards available, in order to carry out metabolization studies, exposure assessment or monitoring modified AFs in food. The aim of this study is to show the recent trends in analytical methods based on LC-HRMS for determination of AFs.

Field Monitoring of Aflatoxins in Feed and Milk of High-Yielding Dairy Cows under Two Feeding Systems

Aflatoxin M1 (AFM1) is a hydroxylated metabolite of aflatoxin B1 (AFB1) that can be excreted in milk of cows after consuming contaminated feed. The aim of this study consisted of a field monitoring to assess the contamination levels of AFB1 in 60 feed samples from two feeding systems for high-yielding dairy cows and of AFM1 in the corresponding raw milk samples. The aflatoxins were analyzed by in-house validated methods based on high-performance liquid chromatography (HPLC) with fluorescence detection. AFB1 was detected in 55% of feed samples (mean 0.61 μg/kg, with 2 samples exceeding the European Union (EU) maximum level set at 5 μg/kg), with greater incidence and concentration in compound feed than in unifeed rations (p < 0.05). AFM1 was detected in 38.3% milk samples (mean 12.6 ng/kg, with 5 samples exceeding the EU maximum level set at 50 ng/kg), with a higher occurrence in milk of cows fed compound feed, as well as in spring milk compared to that produced in winter. The overall transfer ratio of aflatoxins from feed to milk was 3.22%, being higher in cows fed with compound feed and in spring milkings. In a selection of positive matched samples (n = 22), the ratio AFM1/AFB1 exceeded the European Food Safety Authority (EFSA) estimated 6% threshold for high-yielding dairy cows.

Comparison of four immunoassays to an HPLC method for the therapeutic drug monitoring of methotrexate: Influence of the hydroxylated metabolite levels and impact on clinical threshold

Objectives Methotrexate requires therapeutic drug monitoring in oncology because of narrow therapeutic index, especially the metabolite 7-hydroxymethotrexate exhibits nephrotoxicity. The goal of this study was to evaluate different assays and their impact on clinical decisions. Methods Following routine measurement with Abbott TDxFLx® assay (MTX-TDX), 62 samples were analysed on Architect®i1000 (MTX-ARCHI), Xpand® (ARK/XPND), Indiko® (ARK/INDI), and HPLC (MTX-HPLC) as the reference method. The influence of 7-hydroxymethotrexate was explored on ARK reagent to document the cause of the observed bias. ROC curves were built to study the impact of the method on the discharge thresholds for the patients at three levels. Results Total imprecision was below 2.60% for the methotrexate-ARCHI and close to 10% for both ARK assays for plasma pools. The correlation coefficients were 0.93, 0.93, 0.89 and 0.95, the Bland–Altman difference plot revealed a bias of 0.075, 0.037, 0.049 and –0.002, and the number of results exceeding the TE criteria of 0.1 µM was 17 (27%), 13 (21%), 15 (24%) and 15 (24%) for MTX-TDX, ARK/INDI, ARK/XPND and MTX-ARCHI, respectively. Cross reactivity with 7-hydroxymethotrexate was between 1 and 9%. Overestimation of methotrexate concentration was between –4% and +32%. The most robust clinical level was found to be the highest level (0.2 µM) with ROC curves. Conclusions The authors found the best results for imprecision with chemiluminescent microparticle immunoassay method on methotrexate-ARCHI, with bias below to the RICOS recommendations and best correlation to the reference method. Impact on the threshold values for clinical decision need to be clearly exposed to clinicians.

Determination of Aflatoxin M1 in Liquid Milk, Cheese, and Selected Milk Proteins by Automated Online Immunoaffinity Cleanup with Liquid Chromatography‒Fluorescence Detection

Background Aflatoxins are secondary metabolites produced by a number of species of Aspergillus fungi. Aflatoxin M1 (AFM1) is a hydroxylated metabolite of aflatoxin B1 and is found in the milk of cows fed with feed spoilt by Aspergillus species. AFM1 is carcinogenic, especially in the liver and kidneys, and mutagenic, and is also an immunosuppressant in humans. Objective A high-throughput method for the quantitative analysis of AFM1 that is applicable to liquid milk, cheese, milk protein concentrate (MPC), whey protein concentrate (WPC), whey protein isolate (WPI), and whey powder (WP) was developed and validated. Method AFM1 in cheese, milk, and protein products is extracted using 1% acetic acid in acetonitrile with citrate salts. The AFM1 in the resulting extract is concentrated using RIDA®CREST/IMMUNOPREP® ONLINE cartridges followed by quantification by HPLC‒fluorescence. Results The method was shown to be accurate for WP, WPC, WPI, MPC, liquid milk, and cheese, with acceptable recovery (81–112%) from spiked samples. Acceptable precision for WP, WPC, WPI, MPC, liquid milk, and cheese was confirmed, with repeatabilities of 4–12% RSD and intermediate precisions of 5–13% RSD. Method detection limit and ruggedness experiments further demonstrated the suitability of this method for routine compliance testing. An international proficiency scheme (FAPAS) cheese sample showed that this method gave results that were comparable with those from other methods. Conclusions A method for high-throughput, routine testing of AFM1 is described. The method was subjected to single-laboratory validation and was found to be accurate, precise, and fit-for-purpose. Highlights An automated online immunoaffinity cleanup HPLC‒fluorescence method for milk proteins, cheese, and milk was developed and single-laboratory validated. It allows for high-throughput analysis of AFM1 and can be used for the analysis of AFM1 in whey protein products.