Use of a physiologically-based pharmacokinetic model to explore the potential disparity in nicotine disposition between adult and adolescent nonhuman primates.

The widespread use and high abuse liability of tobacco products has received considerable public health attention, in particular for youth, who are vulnerable to nicotine addiction. In this study, adult and adolescent squirrel monkeys were used to evaluate age-related metabolism and pharmacokinetics of nicotine after intravenous administration. A physiologically-based pharmacokinetic (PBPK) model was created to characterize the pharmacokinetic behaviors of nicotine and its metabolites, cotinine, trans-3'-hydroxycotinine (3'-OH cotinine), and trans-3'-hydroxycotinine glucuronide (3'-OH cotinine glucuronide) for both adult and adolescent squirrel monkeys. The PBPK nicotine model was first calibrated for adult squirrel monkeys utilizing in vitro nicotine metabolic data, plasma concentration-time profiles and cumulative urinary excretion data for nicotine and metabolites. Further model refinement was conducted when the calibrated adult model was scaled to the adolescents, because adolescents appeared to clear nicotine and cotinine more rapidly relative to adults. More specifically, the resultant model parameters representing systemic clearance of nicotine and cotinine for adolescent monkeys were approximately two- to three-fold of the adult values on a per body weight basis. The nonhuman primate PBPK model in general captured experimental observations that were used for both model calibration and evaluation, with acceptable performance metrics for precision and bias. The model also identified differences in nicotine pharmacokinetics between adolescent and adult nonhuman primates which might also be present in humans.

Hepatocyte spheroids as a competent in vitro system for drug biotransformation studies: nevirapine as a bioactivation case study.

The development of metabolically competent in vitro models is of utmost importance for predicting adverse drug reactions, thereby preventing attrition-related economical and clinical burdens. Using the antiretroviral drug nevirapine (NVP) as a model, this work aimed to validate rat hepatocyte 3D spheroid cultures as competent in vitro systems to assess drug metabolism and bioactivation. Hepatocyte spheroids were cultured for 12 days in a stirred tank system (3D cultures) and exposed to equimolar dosages of NVP and its two major Phase I metabolites, 12-OH-NVP and 2-OH-NVP. Phase I NVP metabolites were detected in the 3D cultures during the whole culture time in the same relative proportions reported in in vivo studies. Moreover, the modulation of SULT1A1 activity by NVP and 2-OH-NVP was observed for the first time, pointing their synergistic effect as a key factor in the formation of the toxic metabolite (12-sulfoxy-NVP). Covalent adducts formed by reactive NVP metabolites with N-acetyl-L-cysteine and bovine serum albumin were also detected by high-resolution mass spectrometry, providing new evidence on the relative role of the reactive NVP metabolites, 12-sulfoxy-NVP, and NVP quinone methide, in toxicity versus excretion pathways. In conclusion, these results demonstrate the validity of the 3D culture system to evaluate drug bioactivation, enabling the identification of potential biomarkers of bioactivation/toxicity, and providing new evidence to the mechanisms underlying NVP-induced toxic events. This model, integrated with the analytical strategies described herein, is of anticipated usefulness to the pharmaceutical industry, as an upstream methodology for flagging drug safety alerts in early stages of drug development.

Hybrid Quadrupole Mass Filter - Radial Ejection Linear Ion Trap and Intelligent Data Acquisition Enable Highly Multiplex Targeted Proteomics.

Targeted mass spectrometry (MS) methods are powerful tools for selective and sensitive analysis of peptides identified by global discovery experiments. Selected reaction monitoring (SRM) is currently the most widely accepted MS method in the clinic, due to its reliability and analytical performance. However, due to limited throughput and the difficulty in setting up and analyzing large scale assays, SRM and parallel reaction monitoring (PRM) are typically used only for very refined assays of on the order of 100 targets or less. Here we introduce a new MS platform with a quadrupole mass filter, collision cell, linear ion trap architecture that has increased acquisition rates compared to the analogous hardware found in the Orbitrap™ Tribrid™ series instruments. The platform can target more analytes than existing SRM and PRM instruments - in the range of 5000 to 8000 peptides per hour. This capability for high multiplexing is enabled by acquisition rates of 70-100 Hz for peptide applications, and the incorporation of real-time chromatogram alignment that adjusts for retention time drift and enables narrow time scheduled acquisition windows. Finally, we describe a Skyline external software tool that implements the building of targeted methods based on data independent acquisition chromatogram libraries or unscheduled analysis of heavy labeled standards. We show that the platform delivers ~10x lower LOQs than traditional SRM analysis for a highly multiplex assay and also demonstrate how analytical figures of merit change while varying method duration with a constant number of analytes, or by keeping a constant time duration while varying the number of analytes.

A workflow for targeted proteomics assay development using a versatile linear ion trap.

Advances in proteomics and mass spectrometry have enabled the study of limited cell populations, such as single-cell proteomics, where high-mass accuracy instruments are typically required. While triple quadrupoles offer fast and sensitive nominal resolution measurements, these instruments are effectively limited to targeted proteomics. Linear ion traps (LITs) offer a versatile, cost-effective alternative capable of both targeted and global proteomics. We demonstrate a workflow using a newly released, hybrid quadrupole-LIT instrument for developing targeted proteomics assays from global data-independent acquisition (DIA) measurements without needing high-mass accuracy. Gas-phase fraction-based DIA enables rapid target library generation in the same background chemical matrix as each quantitative injection. Using a new software tool embedded within EncyclopeDIA for scheduling parallel reaction monitoring assays, we show consistent quantification across three orders of magnitude of input material. Using this approach, we demonstrate measuring peptide quantitative linearity down to 25x dilution in a background of only a 1 ng proteome without requiring stable isotope labeled standards. At 1 ng total protein on column, we found clear consistency between immune cell populations measured using flow cytometry and immune markers measured using LIT-based proteomics. We believe hybrid quadrupole-LIT instruments represent an economic solution to democratizing mass spectrometry in a wide variety of laboratory settings.

Development of highly multiplex targeted proteomics assays in biofluids using the Stellar mass spectrometer.

The development of targeted assays that monitor biomedically relevant proteins is an important step in bridging discovery experiments to large scale clinical studies. Targeted assays are currently unable to scale to hundreds or thousands of targets. We demonstrate the generation of large-scale assays using a novel hybrid nominal mass instrument. The scale of these assays is achievable with the Stellar™ mass spectrometer through the accommodation of shifting retention times by real-time alignment, while being sensitive and fast enough to handle many concurrent targets. Assays were constructed using precursor information from gas-phase fractionated (GPF) data-independent acquisition (DIA). We demonstrate the ability to schedule methods from an orbitrap and linear ion trap acquired GPF DIA library and compare the quantification of a matrix-matched calibration curve from orbitrap DIA and linear ion trap parallel reaction monitoring (PRM). Two applications of these proposed workflows are shown with a cerebrospinal fluid (CSF) neurodegenerative disease protein PRM assay and with a Mag-Net enriched plasma extracellular vesicle (EV) protein survey PRM assay.

Dose-response assessment of nephrotoxicity from a twenty-eight-day combined-exposure to melamine and cyanuric acid in F344 rats.

The adulteration of pet food with melamine and derivatives, including cyanuric acid, has been implicated in the kidney failure and death of cats and dogs in the USA and other countries. In a previous 7-day dietary study in F344 rats, we established a no-observed-adverse-effect level (NOAEL) for a co-exposure to melamine and cyanuric acid of 8.6 mg/kg bw/day of each compound, and a benchmark dose lower confidence limit (BMDL) of 8.4-10.9 mg/kg bw/day of each compound. To ascertain the role played by the duration of exposure, we treated F344 rats for 28 days. Groups of male and female rats were fed diet containing 0 (control), 30, 60, 120, 180, 240, or 360 ppm of both melamine and cyanuric acid. The lowest dose that produced histopathological alterations in the kidney was 120 ppm, versus 229 ppm in the 7-day study. Wet-mount analysis of kidney sections demonstrated the formation of melamine cyanurate spherulites in one male and two female rats at the 60 ppm dose and in one female rat at the 30 ppm dose, establishing a NOAEL of 2.1mg/kg bw/day for males and <2.6 mg/kg bw/day for females, and BMDL values as low as 1.6 mg/kg bw/day for both sexes. These data demonstrate that the length of exposure is an important component in the threshold of toxicity from a co-exposure to these compounds and suggest that the current risk assessments based on exposures to melamine alone may not reflect sufficiently the risk of a co-exposure to melamine and cyanuric acid.

Effect of urinary pH upon the renal toxicity of melamine and cyanuric acid.

In 2007, dietary exposure to "scrap melamine' resulted in the death of a large number of cats and dogs, which was attributed to the formation of melamine cyanurate crystals in their kidneys. In this study, we investigated if changes in urinary pH could diminish the renal toxicity associated with exposure to combinations of melamine and cyanuric acid. Female Sprague-Dawley rats were treated for three days with suspensions of melamine and cyanuric acid at doses that were expected to induce renal toxicity. Dosing was then discontinued and the rats were treated for seven days with drinking water solutions (i.e., ammonium chloride and sodium bicarbonate) that would alter urinary pH. The urinary pH of rats administered ammonium chloride drinking water decreased from pH 6.0-6.2 to pH 5.1-5.2. This was accompanied by a decrease in the incidence of melamine cyanurate crystals in the kidneys and a decrease in the incidence of renal lesions. These data suggest that acidification of urine may help overcome the renal toxicities associated with the formation of melamine cyanurate crystals in the kidney.

A rapid and highly sensitive UPLC-ESI-MS/MS method for the analysis of the fatty acid profile of edible vegetable oils.

The analysis of the fatty acid profile of triglycerides has long played a central role in the evaluation and classification of edible vegetable oils. However, the range of analytical procedures available to evaluate these profiles remains limited and are typically based on transesterification of the triglyceride fatty acid residues to methyl esters, followed by capillary gas-liquid chromatography (GC) coupled with flame ionization or mass spectrometry detection. Although robust and long-proven, these analytical methods tend to entail long chromatographic runs and are relatively insensitive. In order to expand the range of available techniques for the analysis of the fatty acid profile of triglycerides in vegetable oils, we report herein a novel method based upon a rapid and straightforward transesterification of the triglycerides with dimethylaminoethanol under alkaline conditions, followed by a "dilute-and-shoot" analysis by ultra-performance liquid chromatography coupled with electrospray tandem mass spectrometry. The chromatographic analysis is accomplished in 1.5 min, affording a high throughput of samples compared to techniques based upon GC approaches. The method performance was assessed intra- and inter-day with 10 representative saturated and unsaturated fatty acids ranging from C8 to C18 and afforded fatty acid profile accuracies of 93-108% and imprecisions of only 0.3-2.0%. The limit of quantification of the method, estimated as the minimum amount of derivatized oil sample capable of affording less than 20% accuracy and precision error was determined to be approximately 0.5 pg on-column, making this new method potentially valuable for fields where high sensitivity, precision, and accuracy may be required, such as in toxicology studies, forensics, archeology, or art analysis.

High resolution mass spectrometry-based methodologies for identification of Etravirine bioactivation to reactive metabolites: In vitro and in vivo approaches.

Drug bioactivation to reactive metabolites capable of covalent adduct formation with bionucleophiles is a major cause of drug-induced adverse reactions. Therefore, elucidation of reactive metabolites is essential to unravel the toxicity mechanisms induced by drugs and thereby identify patient subgroups at higher risk. Etravirine (ETR) was the first second-generation Non-Nucleoside Reverse Transcriptase Inhibitor (NNRTI) to be approved, as a therapeutic option for HIV-infected patients who developed resistance to the first-generation NNRTIs. Additionally, ETR came into market aiming to overcome some adverse effects associated with the previously used efavirenz (neurotoxicity) and nevirapine (hepatotoxicity) therapies. Nonetheless, post-marketing reports of severe ETR-induced skin rash and hypersensitivity reactions have prompted the U.S. FDA to issue a safety alert on ETR. Taking into consideration that ETR usage may increase in the near future, due to the possible use of the drug for coinfection with malaria and HIV, the development of reliable prognostic tools for early risk/benefit estimations is urgent. In the current study, high resolution mass spectrometry-based methodologies were integrated with MS3 experiments for the identification of reactive ETR metabolites/adducts: 1) in vitro incubation of the drug with human and rat liver S9 fractions in the presence of Phase I and II co-factors, including glutathione, as a trapping bionucleophile; and 2) in vivo, using urine samples from HIV-infected patients on ETR therapy. We obtained evidence for multiple bioactivation pathways leading to the formation of covalent adducts with glutathione and N-acetyl-L-cysteine. These results suggest that similar reactions may occur with cysteine residues of proteins, supporting a role for ETR bioactivation in the onset of the toxic effects elicited by the drug. Additionally, ETR metabolites stemming from amine oxidation, with potential toxicological significance, were identified in vitro and in vivo. Also noteworthy is the fact that new metabolic conjugation pathways of glucuronide metabolites were demonstrated for the first time, raising questions about their potential toxicological implications. In conclusion, these results represent not only a contribution towards the elucidation of new metabolic pathways of drugs in general but also an important step towards the elucidation of potentially toxic ETR pathways, whose understanding may be crucial for reliable risk/benefit estimations of ETR-based regimens.

Urinary signature of pig carcasses with boar taint by liquid chromatography-high-resolution mass spectrometry.

Boar taint is an offensive odour that can occur while cooking pork or pork products and is identified in some uncastrated male pigs that have reached puberty. It is widely held that boar taint is the result of the accumulation in back fat of two malodorous compounds: androstenone and skatole. The purpose of this study is to assess a mass spectrometry-based metabolomics strategy to investigate the metabolic profile of urine samples from pig carcasses presenting low (untainted) and high (tainted) levels of androstenone and skatole in back fat. Urine samples were analysed by LC-ESI(+)-HRMS. Discrimination between tainted and untainted animals was observed by the application of multivariate statistical analysis, which allowed candidate urinary biomarkers to be highlighted. These urinary metabolites were positively correlated to androstenone and skatole levels in back fat. Therefore, the study suggests that the measurement of these urinary metabolites might provide information with regard to androstenone and skatole levels in live pigs.

New insights into the molecular mechanisms of chemical carcinogenesis: In vivo adduction of histone H2B by a reactive metabolite of the chemical carcinogen furan.

Furan is a rodent hepatocarcinogen ubiquitously found in the environment and heat-processed foods. Furan undergoes cytochrome P450 2E1-catalyzed bioactivation to cis-2-butene-1,4-dial (BDA), which has been shown to form an electrophilic conjugate (GSH-BDA) with glutathione. Both BDA and GSH-BDA yield covalent adducts with lysine residues in proteins. Dose- and time-dependent epigenetic histone alterations have been observed in furan-treated rats. While the covalent modification of histones by chemical carcinogens has long been proposed, histone-carcinogen adducts have eluded detection in vivo. In this study, we investigated if the covalent modification of histones by furan may occur in vivo prior to epigenetic histone alterations. Using a "bottom-up" methodology, involving the analysis of tryptic peptides by liquid chromatography - high resolution mass spectrometry, we obtained evidence for a cross-link between GSH-BDA and lysine 107 of histone H2B isolated from the livers of male F344 rats treated with tumorigenic doses of furan. This cross-link was detected at the shortest treatment period (90 days) in the lowest dose group (0.92mg/kg body weight/day), prior to the identification of epigenetic changes, and occurred at a lysine residue that is a target for epigenetic modifications and crucial for nucleosome stability. Our results represent the first unequivocal proof of the occurrence of carcinogen-modified histones in vivo and suggest that such modification happens at the initial stages of furan-induced carcinogenesis. This type of alteration may be general in scope, opening new insights into the mechanisms of chemical carcinogenesis/toxicity and new opportunities for the development of early compound-specific biomarkers of exposure.

Urinary biomarker detection of melamine- and cyanuric acid-induced kidney injury in rats.

Oral coexposure of rats to melamine (MEL) and cyanuric acid (CYA) results in a dose-dependent increase in the formation of MEL-CYA crystals in the kidney. The aim of this study was to determine if urinary biomarkers of acute kidney injury could be used to noninvasively detect renal damage associated with crystal formation in the kidneys of MEL- and CYA-exposed rats. Urine was obtained on days 0 (predose), 2, 4, 14, and 28 from male and female Fischer 344 rats fed a diet supplemented with 0, 120, 180, or 240 ppm each of MEL and CYA. A number of urinary protein biomarkers (kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, osteopontin, albumin, alpha-GST, GST-Yb1, renal papillary antigen 1 [RPA-1], and clusterin) were measured using a multiplex assay system. The results showed that RPA-1 (distal tubule and collecting duct injury biomarker) was elevated on day 28 at the 120 ppm dose and higher in male rats and at the 180 ppm dose and higher in female rats; however, other urinary protein biomarkers were significantly elevated only at the 240 ppm dose. Significant elevation in blood urea nitrogen and serum creatinine levels, and severe renal damage evidenced by histopathology, were observed after 28 days of exposure to the highest dose, despite the fact that MEL-CYA crystals were observable at the 120 and 180 ppm doses. These data indicate that RPA-1 may serve as a noninvasive urinary biomarker for the detection and monitoring of obstructive nephropathy associated with MEL-CYA exposure.

Pharmacokinetics of melamine and cyanuric acid and their combinations in F344 rats.

The intentional adulteration of pet food with melamine and cyanuric acid has been implicated in the kidney failure and death of a large number of cats and dogs in the United States. Although individually these compounds present low toxicity in a range of experimental animals, coexposure can lead to the formation of melamine cyanurate crystals in the nephrons and eventual kidney failure. Given this mode of action, a good understanding of the pharmacokinetic profiles of melamine and cyanuric acid and their combinations is essential to define properly the risk associated with different exposure scenarios. Previous studies have investigated the individual pharmacokinetic profiles of melamine and cyanuric acid. In this work, we report a comparison between the pharmacokinetic profiles of melamine and cyanuric acid administered individually, administered simultaneously as the individual compounds, and administered as a preformed melamine cyanurate complex. Although the oral coadministration of 1 mg/kg body weight of melamine and cyanuric acid did not alter significantly the pharmacokinetic profiles in relation to those determined upon individual oral administration of each compound, the administration of equal amounts of each triazine as the preformed melamine cyanurate complex significantly altered the pharmacokinetics, with reduced bioavailability of both compounds, lower observed maximum serum concentrations, delayed peak concentrations, and prolonged elimination half lives. These results indicate that in order to estimate properly the combined nephrotoxic potential of melamine and cyanuric acid, the experimental design of toxicological experiments and the evaluation of animal or human exposure scenarios should consider the detailed mode of exposure, with particular emphasis on any possible ex vivo formation of melamine cyanurate.

Low-level quantification of melamine and cyanuric acid in limited samples of rat serum by UPLC-electrospray tandem mass spectrometry.

This paper reports the development and validation of a methodology for the low-level quantification of melamine and cyanuric acid in limited samples of rat serum. The methodology, based upon ion-exchange solid phase extraction (SPE) and ultra-performance liquid chromatography (UPLC) coupled with electrospray tandem mass spectrometry (MS/MS) in multiple reaction monitoring (MRM) mode, relies on the use of stable isotope-labeled internal standards and requires only 15 µL samples of serum. The method provides a recovery of 80-110% of melamine with a signal suppression of ca. 55%, and a recovery of 50-90% of cyanuric acid with a signal suppression ca. 40-60%, affording lower limits of quantification (LLOQ) for melamine or cyanuric acid of, respectively, 5 ppb (mean accuracy 109%; CV=4.9%) and 10 ppb (mean accuracy 96%; CV=8.6%). The small sample requirements, excellent sensitivity, accuracy and precision, and high-throughput (5 min of instrument run time) make this methodology optimal for toxicokinetic or exposure assessments studies.

Dose-response assessment of nephrotoxicity from a 7-day combined exposure to melamine and cyanuric acid in F344 rats.

The intentional adulteration of pet food with melamine and derivatives, including cyanuric acid, has been implicated in the kidney failure and death of a large number of cats and dogs in the United States. Although individually these compounds present low toxicity, coexposure can lead to the formation of melamine cyanurate crystals in the nephrons and eventual kidney failure. To determine the dose-response for nephrotoxicity upon coadministration of melamine and cyanuric acid, groups of male and female F344 rats (six animals per sex per group) were fed 0 (control), 7, 23, 69, 229, or 694 ppm of both melamine and cyanuric acid; 1388 ppm melamine; or 1388 ppm cyanuric acid in the diet for 7 days. No toxicity was observed in the rats exposed to the individual compounds, whereas anorexia and a statistically significant increase in blood urea nitrogen and serum creatinine levels was observed in the animals treated with 229 and 694 ppm melamine and cyanuric acid. The kidneys of these animals were grossly enlarged and pale yellow. Large numbers of crystalline structures deposited in the tubules were seen on sections in kidneys from all rats in these treatment groups. No significant changes were detected in the remaining treatment groups exposed to both melamine and cyanuric acid. In the melamine-only treatment group, 5 of 12 rats had scattered crystals present in renal tubules when examined by wet mount. These were not observed by histopathology. The observed adverse effect level (8.6 mg/kg bw [body weight]/day) and benchmark dose modeling data (8.4-10.9 mg/kg bw/day) determined in this study suggest that the tolerable daily intake values derived from studies conducted with melamine alone may underestimate the risk from coexposures to melamine and cyanuric acid.