Metabolite Exposure Research Articles

Population pharmacokinetic and pharmacodynamic modeling of capecitabine and its metabolites in breast cancer patients.

The present study was performed to examine relationships between systemic exposure of capecitabine metabolites (5-FU, 5'-DFCR and 5'-DFUR) and toxicity or clinical response in patients with metastatic breast cancer. A population pharmacokinetic model for capecitabine and its three metabolites was built. Typical parameter values, characteristics of random distributions, associated with parameters, and covariates impact were estimated. Area under the curve (AUC) were computed for 5-FU and compared with grades of toxicity. Pharmacokinetic modeling was based on data collected on the first treatment cycle. Toxicity was assessed on the two first treatment cycles. The study was conducted in 43 patients. The population pharmacokinetic model (a one-compartment model per compound) was able to capture the very complex absorption process of capecitabine. Statistically significant covariates were cytidine deaminase, alkaline phosphatase and dihydrouracilemia (UH2)/uracilemia (U) ratio. UH2/U ratio was the most significant covariate on 5-FU elimination and CDA on the transformation of 5'-DFCR in 5'-DFUR. A trend was observed between 5-FU AUC and thrombopenia toxicity grades, but not with other toxicities. Best clinical response was not linked to systemic exposure of capecitabine metabolites. In our study, we propose a model able to describe, meanwhile, and its main metabolites, with a complex absorption process and inclusion of enzyme activity covariates such as CDA and UH2/U ratio. Trial registration Eudract 2008-004136-20, 2008/11/26.

Metabolic disposition of [14 C]-abivertinib, an epidermal growth factor receptor tyrosine kinase inhibitor: Role of glutathione conjugation.

To determine the absorption, distribution, metabolism and excretion of abivertinib, a third-generation epidermal growth factor receptor tyrosine kinase inhibitor, in patients with advanced non-small cell lung cancer (NSCLC). Seven patients with advanced NSCLC were given a single 200 mg/83 μCi oral suspension of [14 C]-abivertinib. Blood, urine and faeces were collected. Mass balance of radioactivity, the pharmacokinetics of abivertinib, and the total radioactivity were determined. Metabolite profiling and characterisation were performed. The mean recovery was 82.16%, with 2.38 and 79.78% of the radioactive dose excreted in urine and faeces, respectively. The unchanged abivertinib was the major radioactive component detected in plasma within the first 24 hours after dosing, accounting for 59.17% of the total drug-related radioactivity. Abivertinib in urine accounted for only 0.96% of the administered dose, whereas in faeces it accounted for 33.36%. Eight metabolites were detected and characterised in plasma, among which MII-7, a product of cysteine glycine conjugate, was the only circulating metabolite, accounting for approximate 10.6% of the total drug-related exposure. MII-2 (an abivertinib cysteine-glycine adduct) and M7 (a reduced product of abivertinib) were the 2 major metabolites in the excreta, accounting for 20.0 and 12.4%, respectively, of the drug-related radioactivity in faeces. Following a single oral administration, the unchanged abivertinib was the predominant drug-related material in plasma, urine and faeces. The drug-related materials were primarily eliminated via the faecal route. Direct glutathione conjugation of abivertinib played a significant role in the metabolic clearance and metabolite exposure of abivertinib.

Longitudinal exposure to pyrethroids 3-PBA, trans-TCC & 2,4 D herbicides in rural schoolchildren, Maule, Chile.

Background: Several studies show that early exposure to pesticides and during the preschool and school stage affects the development and health of children. In Maule, a series of studies have shown the exposure of organophosphate pesticides(OP), however, there are no studies assessing exposure to pyrethroids and herbicide 2,4 D. Objetive: To evaluate exposure to pyrethroids 3-phenoxybenzoic acid 3-PBA, trans-2,2-(dichloro)-2-dimethylvinylcyclopropane carboxylic acid trans-DCCA(Trans-TCC) and 2,4 D herbicides in schoolchildren. Method: Longitudinal study, 48 schoolchildren from 5 to 13 years old from two rural schools in the Maule. Specific metabolites of pyrethroids 3-PBA, Trans-TCC and 2.4 D in the urine of the school children were evaluated. The data were correlated with an earlier study that evaluated the exposure of OP metabolites. Two samples were taken in September and 2 samples in November, a total of 384 urine samples were obtained during 2016 and 2017. Data analysis: descriptive, U Mann Whitney for repeated measurements, Spearman and GEE. Results: The 100% of schoolchildren have more than two pesticide metabolites in their urine in both years, with the 3-PBA metabolite being the most frequent. 3-PBA in November 2017 increases its presence in urine compared to 2016(from 0.69 μg/L in 2016 to 1.90μg/L in 2017). In 2016, the specific metabolites of 3-PBA correlate with TCC, 2,4 D, chlorpyrifos, diazinon, paration. In 2017, 3-PBA was correlated with 2,4 D and TCC. Importantly, the concentrations of 3-PBA pesticide metabolites in urine found in Chilean children was higher than studies in the USA where there is an association of exposure from prenatal stage to these metabolites with difficulties in emotional control and attention. Conclusions: Promote the monitoring, training and active epidemiological surveillance of agricultural communities. The increased use of pyrethroids is worrying. Promote the intervention of school communities, involving government agencies and agricultural companies, considering preventing to the population.

Pressure regulated basis for gene transcription by delta-cell micro-compliance modeled in silico: Biphenyl, bisphenol and small molecule ligand models of cell contraction-expansion.

Molecular diameter, lipophilicity and hydrophilicity exclusion affinity limits exist for small molecule carrier-mediated diffusion or transport through channel pores or interaction with the cell surface glycocalyx. The molecular structure lipophilicity limit for non-specific carrier-mediated transmembrane diffusion through polarity-selective transport channels of the cell membrane is Lexternal structure ∙ Hpolar group-1 of ≥ 1.07. The cell membrane channel pore size is > 0.752 and < 0.758 nm based on a 3-D ellipsoid model (biphenyl), and within the molecular diameter size range 0.744 and 0.762 nm based on a 2-D elliptical model (alkanol). The adjusted van der Waals diameter (vdWD, adj; nm) for the subset of halogenated vapors is predictive of the required MAC for anesthetic potency at an initial (-) Δ Cmicro effect. The molecular structure L ∙ Hpolar group-1 for Neu5Ac is 0.080, and the L ∙ Hpolar group-1 interval range for the cell surface glycocalyx hydrophilicity barrier interaction is 0.101 (Saxitoxin, Stx; Linternal structure ∙ Hpolar group-1) - 0.092 (m-xylenediamine, Lexternal structure · Hpolar group). Differential predictive effective pressure mapping of gene activation or repression reveals that p-dioxin exposure results in activation of AhR-Erβ (Arnt)/Nrf-2, Pparδ, Errγ (LxRα), Dio3 (Dio2) and Trα limbs, and due to high affinity Dio2 and Dio3 (OH-TriCDD, Lext · H-1: 1.91–4.31) exothermy-antagonism (Δ contraction) with high affinity T4/rT3-TRα-mediated agonism (Δ expansion). co-planar PCB metabolite exposure (Lext · H-1: 1.95–3.91) results in activation of AhR (Erα/β)/Nrf2, Rev-Erbβ, Errα, Dio3 (Dio2) and Trα limbs with a Δ Cmicro contraction of 0.89 and Δ Cmicro expansion of 1.05 as compared to p-dioxin. co-, ortho-planar PCB metabolite exposure results in activation of Car/PxR, Pparα (Srebf1,—Lxrβ), Arnt (AhR-Erβ), AR, Dio1 (Dio2) and Trβ limbs with a Δ Cmicro contraction of 0.73 and Δ Cmicro expansion of 1.18 (as compared to p-dioxin). Bisphenol A exposure (Lext struct ∙ H-1: 1.08–1.12, BPA–BPE, Errγ; BPAF, Lext struct ∙ H-1: 1.23, CM Erα, β) results in increased duration at Peff for Timm8b (Peff 0.247) transcription and in indirect activation of the AhR/Nrf-2 hybrid pathway with decreased duration at Peff 0.200 (Nrf1) and increased duration at Peff 0.257 (Dffa). The Bpa/Bpaf convergent pathway Cmicro contraction-expansion response increase in the lower Peff interval is 0.040; in comparison, small molecule hormone Δ Cmicro contraction-expansion response increases in the lower Peff intervals for gene expression ≤ 0.168 (Dex· GR) ≥ 0.156 (Dht · AR), with grade of duration at Peff (min·count) of 1.33x105 (Dex/Cort) and 1.8–2.53x105 (Dht/R1881) as compared to the (-) coupled (+) Δ Cmicro Peff to 0.136 (Wnt5a, Esr2) with applied DES (1.86x106). The subtype of trans-differentiated cell as a result of an applied toxin or toxicant is predictable by delta-Cmicro determined by Peff mapping. Study findings offer additional perspective on the basis for pressure regulated gene transcription by alterations in cell micro-compliance (Δ contraction-expansion, Cmicro), and are applicable for the further predictive modeling of gene to gene transcription interactions, and small molecule modulation of cell effective pressure (Peff) and its potential.

Physiologically Based Pharmacokinetic Modeling of Transdermal Selegiline and Its Metabolites for the Evaluation of Disposition Differences between Healthy and Special Populations.

A physiologically based pharmacokinetic (PBPK) model of selegiline (SEL), and its metabolites, was developed in silico to evaluate the disposition differences between healthy and special populations. SEL is metabolized to methamphetamine (MAP) and desmethyl selegiline (DMS) by several CYP enzymes. CYP2D6 metabolizes the conversion of MAP to amphetamine (AMP), while CYP2B6 and CYP3A4 predominantly mediate the conversion of DMS to AMP. The overall prediction error in simulated PK, using the developed PBPK model, was within 0.5–1.5-fold after intravenous and transdermal dosing in healthy and elderly populations. Simulation results generated in the special populations demonstrated that a decrease in cardiac output is a potential covariate that affects the SEL exposure in renally impaired (RI) and hepatic impaired (HI) subjects. A decrease in CYP2D6 levels increased the systemic exposure of MAP. DMS exposure increased due to a reduction in the abundance of CYP2B6 and CYP3A4 in RI and HI subjects. In addition, an increase in the exposure of the primary metabolites decreased the exposure of AMP. No significant difference between the adult and adolescent populations, in terms of PK, were observed. The current PBPK model predictions indicate that subjects with HI or RI may require closer clinical monitoring to identify any untoward effects associated with the administration of transdermal SEL patch.

The internal exposure of phthalate metabolites and bisphenols in waste incineration plant workers and the associated health risks

Many hazardous substances can be released during incineration of municipal solid waste (MSW), which pose a potential threat to human health. As additives, phthalates (PAEs) and bisphenols (BPs), which are widely used in daily goods, are likely to be present in the released hazardous substances. In the present study, we investigated the urinary levels of phthalate metabolites (mPAEs) and BPs in workers in an MSW incineration plant (the exposed group) and in residents 8 km away (the control group) in Shenzhen, China. The results showed that the median total urinary concentration of mPAEs in workers was significantly higher than that in residents (1.02 × 103 vs. 375 ng/mL). However, there was no significant difference between workers and residents for BPs. Among the mPAEs measured, the most abundant compound was mono-n-butyl phthalate in both exposed and control groups. Monoethyl phthalate and monomethyl phthalate might be potential markers for MSW incineration because of significantly high levels in the exposed group. The workers engaged in different types of workshops showed no significant differences in the urinary levels of mPAEs, also for BPs. It was worth noting that 70.8% of workers were at risk of the non-carcinogenic effects caused by PAEs with diethylhexyl phthalate having the highest risk. Actions should be taken to reduce the risks caused by these hazardous chemicals.

Type 2 diabetes mellitus decreases systemic exposure of clopidogrel active metabolite through upregulation of P-glycoprotein in rats

Patients with diabetic mellitus tend to have a poor response to clopidogrel (Clop) due to reduced generation of active metabolite (Clop-AM). However, the underlying mechanism is not elucidated. A type 2 diabetic mellitus (T2DM) rat model was established by combining high-fat diet feeding and low-dose streptozotocin (STZ) injection. The reduced Clop-AM exposure was observed in T2DM rats after oral administration of Clop. However, in vitro liver microsomes incubated with Clop exhibited increased Clop-AM levels in T2DM rats due to a significant decrease in carboxylesterase (CES)1 expression and activity and a significant increase in the expression or activity of CYP1A2 and CYP3A. Interestingly, different from oral administration, the significantly increased Cmax of Clop-AM was observed in T2DM rats after intravenous injection, with no difference in AUC0-t and t1/2 values between the two strains. Meanwhile, in situ single -pass intestinal perfusion study showed lower absorption rate constant (Ka) and effective apparent permeability values (Peff) of Clop in T2DM rats than in control rats. It is explained by the increased expression or function of P-glycoprotein (P-gp) and pregnane X receptor (PXR) in duodenum and jejunum of T2DM rats. Moreover, the decreased Clop-AM level in T2DM rats was eliminated by the pretreatment of cyclosporin A, a P-gp inhibitor. It suggests that intestinal absorption, not hepatic metabolism is responsible for the reduced Clop-AM exposure in T2DM rats. P-gp might be the key factor causing the reduction of Clop absorption, consequently making less Clop available for Clop-AM formation.

Comparison of aggregated exposure to di(2-ethylhexyl) phthalate from diet and personal care products with urinary concentrations of metabolites using a PBPK model – Results from the Norwegian biomonitoring study in EuroMix

Phthalates are widely used as plasticisers in flexible plastics and containers for food and personal care products (PCPs) and contaminates foods and PCPs. A human biomonitoring (BM) study was performed to study exposure of chemicals from foods and PCPs. For two 24-h periods, adult volunteers (n = 144) in Norway kept diaries on food eaten and usage of PCPs, and collected 24-h urine. Aggregated exposure to di(2-ethylhexyl) phthalate (DEHP) from dietary and PCPs was estimated by Monte-Carlo simulation using Oracle Crystal Ball©. Simulated urinary concentrations using physiologically based pharmacokinetic (PBPK) models were compared with measured urinary metabolites of DEHP, mono-2-ethylhexyl phthalate (MEHP), mono-2-ethyl-5-hydroxyhexyl phthalate (MEHHP), mono-2-ethyl-5-oxohexyl phthalate (MEOHP) and mono-2-ethyl 5-carboxypentyl phthalate (MECCP). DEHP exposure from food are approximately 10 times higher than exposure than from PCPs. The main contributors to dietary exposure are dairy, grain, fruits and vegetables, meat and fish. Body lotion contribute most to the exposure of DEHP from PCPs. Forward-dosimetry gives good convergence with 24-h urinary concentrations of simulated and measured BM data. The measured concentration of the MECCP metabolite correlated well with simulated high exposure, while the measured concentrations of MEHP, MEHHP and MEOHP partly overlapped with both simulated low, medium and high metabolite exposure.

Impact of Plasma Exposure of Statins and Their Metabolites With Major Adverse Cardiovascular Events in Chinese Patients With Coronary Artery Disease.

The selection of optimum statin intensity is inconclusive, and the association of plasma exposure of statins and metabolites with major adverse cardiovascular events (MACEs) is unclear. This study sought to compare the effect of low (quartile 1), intermediate (quartiles 2 and 3), and high (quartile 4) plasma exposure of statins and metabolites on MACE, re-ischemia events and death in patients with coronary artery disease (CAD) at 5 years. A total of 1,644 patients in atorvastatin (AT) cohort and 804 patients in rosuvastatin (RST) cohort were included, and their plasma concentration of statins and metabolites was categorized as low-, mid-, or high-group. The association between the plasma levels of statins and metabolites and the incidence of primary endpoint in patients was assessed by Cox proportional hazard models. Intensive AT exposure (Q4 > 5.32 ng/ml) was significantly associated with increased risk of death compared with low (hazard ratio [HR]: 1.522; 95% confidence interval [CI]: 1.035–1.061; P = 0.0022) or moderate exposure (HR: 2.054; 95% CI: 1.348–3.130; P = 0.0008). This association was also found in AT’s five metabolites (all P < 0.01). In patients with RST treatment, moderate RST concentration (0.53–4.29 ng/ml) versus low concentration had a significantly lower risk of MACE and re-ischemia events. (HR: 0.532, 95% CI: 0.347–0.815, P = 0.0061 and HR: 0.505, 95% CI: 0.310–0.823, P = 0.0061, respectively). A higher plasma exposure of AT and metabolites has a significantly higher risk of death, and moderate RST exposure has a significantly lower risk of MACE and re-ischemia events in Chinese patients with CAD. The harms of high plasma exposure should be considered when prescribing statins to patients because it may be a risk factor for having poor prognosis in patients with CAD.

Effects of Tenofovir on the Single-Dose Pharmacokinetics of Intravenous Morinidazole in Healthy Chinese Subjects.

The effects of multiple-dose administration of tenofovir disoproxil fumarate (TDF) on the pharmacokinetics of morinidazole (MOR) were compared in healthy subjects. MOR exposure was similar, with an area under the curve from 0 h to infinity (AUC0-∞) treatment ratio for MOR+TDF/MOR of 1.01 (90% confidence interval, 0.97 to 1.06). No relevant differences were observed regarding plasma exposure of metabolites. Renal clearances of MOR and its metabolites were not affected by TDF. No unexpected safety or tolerability issues were observed.

Mechanistic PBPK Modeling of Urine pH Effect on Renal and Systemic Disposition of Methamphetamine and Amphetamine.

The effect of urine pH on renal excretion and systemic disposition has been observed for many drugs and metabolites. When urine pH is altered, tubular ionization, passive reabsorption, renal clearance, and systemic exposure of drugs and metabolites may all change dramatically, raising clinically significant concerns. Surprisingly, the urine pH effect on drug disposition is not routinely explored in humans, and regulatory agencies have neither developed guidance on this issue nor required industry to conduct pertinent human trials. In this study, we hypothesized that physiologically based pharmacokinetic (PBPK) modeling could be used as a cost-effective method to examine potential urine pH effect on drug and metabolite disposition. Our previously developed and verified mechanistic kidney model was integrated with a full-body PBPK model to simulate renal clearance and area under the plasma concentration-time curve (AUC) with varying urine pH statuses using methamphetamine and amphetamine as model compounds. We first developed and verified drug models for methamphetamine and amphetamine under normal urine pH condition [absolute average fold error (AAFE) < 1.25 at study level]. Then, acidic and alkaline urine scenarios were simulated. Our simulation results show that the renal excretion and plasma concentration-time profiles for methamphetamine and amphetamine could be recapitulated under different urine pH (AAFE < 2 at individual level). The methamphetamine-amphetamine parent-metabolite full-body PBPK model also successfully simulated amphetamine plasma concentration-time profiles (AAFE < 1.25 at study level) and amphetamine/methamphetamine urinary concentration ratios (AAFE < 2 at individual level) after dosing methamphetamine. This demonstrates that our mechanistic PBPK model can predict urine pH effect on systemic and urinary disposition of drugs and metabolites. SIGNIFICANCE STATEMENT: Our study shows that integrating mechanistic kidney model with full-body physiologically based pharmacokinetic model can predict the magnitude of alteration in renal excretion and area under the plasma concentration-time curve (AUC) of drugs and metabolites when urine pH is changed. This provides a cost-effective method to evaluate the likelihood of renal and systemic disposition changes due to varying urine pH. This is important because multiple drugs and diseases can alter urine pH, leading to quantitatively and clinically significant changes in drug and metabolite disposition that may require adjustment of therapy.

Predicting Clinical Effects of CYP3A4 Modulators on Abemaciclib and Active Metabolites Exposure Using Physiologically Based Pharmacokinetic Modeling.

Abemaciclib, a selective inhibitor of cyclin‐dependent kinases 4 and 6, is metabolized mainly by cytochrome P450 (CYP)3A4. Clinical studies were performed to assess the impact of strong inhibitor (clarithromycin) and inducer (rifampin) on the exposure of abemaciclib and active metabolites. A physiologically based pharmacokinetic (PBPK) model incorporating the metabolites was developed to predict the effect of other strong and moderate CYP3A4 inhibitors and inducers. Clarithromycin increased the area under the plasma concentration‐time curve (AUC) of abemaciclib and potency‐adjusted unbound active species 3.4‐fold and 2.5‐fold, respectively. Rifampin decreased corresponding exposures 95% and 77%, respectively. These changes influenced the fraction metabolized via CYP3A4 in the model. An absolute bioavailability study informed the hepatic and gastric availability. In vitro data and a human radiolabel study determined the fraction and rate of formation of the active metabolites as well as absorption‐related parameters. The predicted AUC ratios of potency‐adjusted unbound active species with rifampin and clarithromycin were within 0.7‐ and 1.25‐fold of those observed. The PBPK model predicted 3.78‐ and 7.15‐fold increases in the AUC of the potency‐adjusted unbound active species with strong CYP3A4 inhibitors itraconazole and ketoconazole, respectively; and 1.62‐ and 2.37‐fold increases with the concomitant use of moderate CYP3A4 inhibitors verapamil and diltiazem, respectively. The model predicted modafinil, bosentan, and efavirenz would decrease the AUC of the potency‐adjusted unbound active species by 29%, 42%, and 52%, respectively. The current PBPK model, which considers changes in unbound potency‐adjusted active species, can be used to inform dosing recommendations when abemaciclib is coadministered with CYP3A4 perpetrators.

Drug-drug interaction (DDI) potential of oral napabucasin in healthy adults.

142 Background: Napabucasin is an NQO1-bioactivatable investigational agent hypothesized to affect multiple oncogenic cellular pathways including pSTAT3 through the generation of reactive oxygen species. This phase 1 open-label study evaluated the DDI potential of napabucasin and its major metabolite (M1) with respect to 7 major human drug cytochrome P450 (CYP) enzymes and the breast cancer resistance protein (BCRP) transporter. Methods: Healthy adult subjects who initially demonstrated they could tolerate administration of 240 or 480 mg twice daily (BID) napabucasin over 2-days (D) received single doses of the CYP and transporter substrates, followed by ≥7-day washout. In the DDI portion, subjects received napabucasin 240 mg BID on D1–11 with the phenotyping cocktail administered on D6 (omeprazole [CYP2C19] 20 mg, caffeine [CYP1A2] 100 mg, flurbiprofen [CYP2C9] 50 mg, bupropion [CYP2B6] 150 mg, dextromethorphan [CYP2D6] 30 mg, and oral midazolam [CYP3A] 2 mg), intravenous (IV) midazolam 2 mg on D7, repaglinide (CYP2C8) 0.25 mg on D8, and rosuvastatin (BRCP transporter) 10 mg on D9. Results: DDI potential was evaluated in 17 subjects. Exposure to omeprazole, flurbiprofen, and oral midazolam with (test) or without (reference) napabucasin 240 mg BID were similar. Napabucasin increased exposure (area under the curve to infinity) to caffeine (124%), IV midazolam (118%), repaglinide (127%), and rosuvastatin (213%); and decreased exposure to bupropion (79%) and dextromethorphan (71%). None of these changes were expected to be clinically meaningful. The exposure of the major metabolites of the probe drugs with or without napabucasin or M1 were similar. Of the 17 subjects, 12 (70.6%) reported adverse events (AEs); 58.8% reported gastrointestinal disorders. One patient had a grade 3 AE (neutrophil count low); no serious AEs were observed. Conclusions: The data suggest minimal in vivo DDI potential for napabucasin with respect to 7 major human drug CYP enzymes and the BCRP transporter. Napabucasin 240 mg BID was generally tolerable in healthy subjects. Co-administration of napabucasin with CYP and transporter substrates was safe and tolerable. Clinical trial information: NCT03411122.

Route of Oseltamivir Administration Affects Metabolite Concentrations in Critically Ill Children.

We performed a prospective cohort study to investigate oseltamivir administration in critically ill children. We found that enteric tube administration of oseltamivir resulted in lower concentrations of its active metabolite compared with oral delivery. These findings could have significant clinical implications, and more studies are required to better understand the effects of administration route on potential lower systemic metabolite exposure.

Increased heart fibrosis and acute infection in a murine Chagas disease model associated with organophosphorus pesticide metabolite exposure

Some reports suggest that exposure to organophosphorus (OP) pesticides increases the incidence of infections. Ethylated dialkylphosphates (EtDAPs) are metabolites of OP pesticides widely distributed with immunomodulatory potential. Chagas disease is produced by Trypanosoma cruzi parasites, and resolution of this infection requires the activation of inflammatory macrophages (MΦ), which results in cardiac fibrosis. Some reports indicate that EtDAPs increase the amount of the anti-inflammatory alternatively activated MΦ (M2; CD206+F4/80+). Therefore, we analyzed the course of T. cruzi infection, MΦ profiles from peritoneal exudate cells (PECs), inflammatory cell infiltration and fibrosis in the heart of BALB/c mice exposed to diethyldithiophosphate (DEDTP), diethylthiophosphate (DETP) or diethylphosphate (DEP, 0.01 g/kg), common DAPs produced by OP pesticides, 24 h before infection with T. cruzi. We found that DEDTP increased the parasite burden in blood by 99% at the peak of the infection and enhanced the myocardial damage due to an increase in infiltrated inflammatory cells (induced by DEDTP or DETP) and fibrosis (induced by EtDAPs). In the PECs, exposure to EtDAPs increased the proportion of the MΦ subpopulations of M2a, M2b and M2d, which are associated with tissue repair. These results indicate that exposure to EtDAPs can exacerbate the acute phase of a parasitic infection and increase the long-term damage to the heart.

Early acetaminophen-protein adducts predict hepatotoxicity following overdose (ATOM-5)

Acetaminophen-protein adducts are specific biomarkers of toxic acetaminophen (paracetamol) metabolite exposure. In patients with hepatotoxicity (alanine aminotransferase [ALT] >1,000 U/L), an adduct concentration ≥1.0 nmol/ml is sensitive and specific for identifying cases secondary to acetaminophen. Our aim was to characterise acetaminophen-protein adduct concentrations in patients following acetaminophen overdose and determine if they predict toxicity. We performed a multicentre prospective observational study, recruiting patients 14 years of age or older with acetaminophen overdose regardless of intent or formulation. Three serum samples were obtained within the first 24 h of presentation and analysed for acetaminophen-protein adducts. Acetaminophen-protein adduct concentrations were compared to ALT and other indicators of toxicity. Of the 240 patients who participated, 204 (85%) presented following acute ingestions, with a median ingested dose of 20 g (IQR 10-40), and 228 (95%) were treated with intravenous acetylcysteine at a median time of 6 h (IQR 3.5-10.5) post-ingestion. Thirty-six (15%) patients developed hepatotoxicity, of whom 22 had an ALT ≤1,000 U/L at the time of initial acetaminophen-protein adduct measurement. Those who developed hepatotoxicity had a higher initial acetaminophen-protein adduct concentration compared to those who did not, 1.63 nmol/ml (IQR 0.76-2.02, n= 22) vs. 0.26 nmol/ml (IQR 0.15-0.41; n= 204; p <0.0001), respectively. The AUROC for hepatotoxicity was 0.98 (95% CI 0.96-1.00; n= 226; p <0.0001) with acetaminophen-protein adduct concentration and 0.89 (95% CI 0.82-0.96; n= 219; p <0.0001) with ALT. An acetaminophen-protein adduct concentration of 0.58 nmol/ml was 100% sensitive and 91% specific for identifying patients with an initial ALT ≤1,000 U/L who would develop hepatotoxicity. Adding acetaminophen-protein adduct concentrations to risk prediction models improved prediction of hepatotoxicity to a level similar to that obtained by more complex models. Acetaminophen-protein adduct concentration on presentation predicted which patients with acetaminophen overdose subsequently developed hepatotoxicity, regardless of time of ingestion. An adduct threshold of 0.58 nmol/L was required for optimal prediction. Acetaminophen poisoning is one of the most common causes of liver injury. This study examined a new biomarker of acetaminophen toxicity, which measures the amount of toxic metabolite exposure called acetaminophen-protein adduct. We found that those who developed liver injury had a higher initial level of acetaminophen-protein adducts than those who did not. Australian Toxicology Monitoring (ATOM) Study-Australian Paracetamol Project: ACTRN12612001240831 (ANZCTR) Date of registration: 23/11/2012.

Maternal and Fetal Blood Pharmacokinetics and Organ Distribution of Atrazine, Propazine, Simazine, and Their Metabolites in Pregnant Rats After Chronic Oral Administration.

Environmental contamination by chlorotriazines has been evidenced in mother-child cohort suggesting more detailed risk assessment of these compounds in drinking water. Exposure of rodents to atrazine (ATZ) has been associated with alterations of endocrine and reproductive functions by disrupting neuroendocrine control at hypothalamus level. Perinatal exposure to low doses of ATZ has been associated with reproductive dysfunction, and to behavioral abnormalities in adult exposed during embryogenesis. The objectives of the current investigation were to (1) evaluate the influence of physicochemical properties of chlorotriazines on tissue distribution in pregnant rats and in fetuses and (2) gain a better understanding of fetal distribution of chlorotriazines in specific tissues, particularly in brain. Serial blood samples were obtained from pregnant rats after administration of ATZ, propazine (PRO), and simazine (SIM) via oral route at a dose of 10 mg/kg from day 15 to day 19. Maternal and fetal tissues were harvested at day 20, 24 h after the last dosing. The metabolic extraction ratio was estimated to 87% suggesting a significant first-pass effect explaining the low oral bioavailability. Blood exposure to parent compounds (ATZ, PRO, and SIM) was negligible (lower than 5%) compared with metabolite exposure. The main metabolite exposure involved diamino-s-chlorotriazine, ranging from 60% to 90% depending on the molecules administered. A correlation between tissue-to-blood ratio and physicochemical descriptors was observed for fat and mammary gland tissues but not for brain in adult rats. A more pronounced distribution in fetal brain was observed for ATZ and PRO, the 2 most lipophilic compounds.

Offline two-dimensional liquid chromatography coupled with ion mobility-quadrupole time-of-flight mass spectrometry enabling four-dimensional separation and characterization of the multicomponents from white ginseng and red ginseng

Inherent complexity of plant metabolites necessitates the use of multi-dimensional information to accomplish comprehensive profiling and confirmative identification. A dimension-enhanced strategy, by offline two-dimensional liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (2D-LC/IM-QTOF-MS) enabling four-dimensional separations (2D-LC, IM, and MS), is proposed. In combination with in-house database-driven automated peak annotation, this strategy was utilized to characterize ginsenosides simultaneously from white ginseng (WG) and red ginseng (RG). An offline 2D-LC system configuring an Xbridge Amide column and an HSS T3 column showed orthogonality 0.76 in the resolution of ginsenosides. Ginsenoside analysis was performed by data-independent high-definition MSE (HDMSE) in the negative ESI mode on a Vion™ IMS-QTOF hybrid high-resolution mass spectrometer, which could better resolve ginsenosides than MSE and directly give the CCS information. An in-house ginsenoside database recording 504 known ginsenosides and 58 reference compounds, was established to assist the identification of ginsenosides. Streamlined workflows, by applying UNIFI™ to automatedly annotate the HDMSE data, were proposed. We could separate and characterize 323 ginsenosides (including 286 from WG and 306 from RG), and 125 thereof may have not been isolated from the Panax genus. The established 2D-LC/IM-QTOF-HDMSE approach could also act as a magnifier to probe differentiated components between WG and RG. Compared with conventional approaches, this dimension-enhanced strategy could better resolve coeluting herbal components and more efficiently, more reliably identify the multicomponents, which, we believe, offers more possibilities for the systematic exposure and confirmative identification of plant metabolites.

Mass balance and metabolite profiling of 14C-guadecitabine in patients with advanced cancer

SummaryPurpose The objective of this mass balance trial was to determine the excretory pathways and metabolic profile of the novel anticancer agent guadecitabine in humans after administration of a 14C-radiolabeled dose of guadecitabine. Experimental design Included patients received at least one cycle of 45 mg/m2 guadecitabine subcutaneously as once-daily doses on Days 1 to 5 of a 28-day cycle, of which the 5th (last) dose in the first cycle was spiked with 14C-radiolabeled guadecitabine. Using different mass spectrometric techniques in combination with off-line liquid scintillation counting, the exposure and excretion of 14C-guadecitabine and metabolites in the systemic circulation, excreta, and intracellular target site were established. Results Five patients were enrolled in the mass balance trial. 14C-guadecitabine radioactivity was rapidly and almost exclusively excreted in urine, with an average amount of radioactivity recovered of 90.2%. After uptake in the systemic circulation, guadecitabine was converted into ß-decitabine (active anomer), and from ß-decitabine into the presumably inactive metabolites M1-M5. All identified metabolites in plasma and urine were ß-decitabine related products, suggesting almost complete conversion via cleavage of the phosphodiester bond between ß-decitabine and deoxyguanosine prior to further elimination. ß-decitabine enters the intracellular activation pathway, leading to detectable ß-decitabine-triphosphate and DNA incorporated ß-decitabine levels in peripheral blood mononuclear cells, providing confirmation that the drug reaches its DNA target site. Conclusion The metabolic and excretory pathways of guadecitabine and its metabolites were successfully characterized after subcutaneous guadecitabine administration in cancer patients. These data support the clinical evaluation of safety and efficacy of the subcutaneous guadecitabine drug product.

Genomics and LC-MS Reveal Diverse Active Secondary Metabolites in Bacillus amyloliquefaciens WS-8.

Bacillus amyloliquefaciens is an important plant disease-preventing and growth-promoting microorganism. B. amyloliquefaciens WS-8 can stimulate plant growth and has strong antifungal properties. In this study, we sequenced the complete genome of B. amyloliquefaciens WS-8 by Pacific Biosciences RSII (PacBio) Single Molecule Real-Time (SMRT) sequencing. The genome consists of one chromosome (3,929,787 bp) and no additional plasmids. The main bacteriostatic substances were determined by genome, transcriptome, and mass spectrometry data. We thereby laid a theoretical foundation for the utilization of the strain. By genomic analysis, we identified 19 putative biosynthetic gene clusters for secondary metabolites, most of which are potentially involved in the biosynthesis of numerous bioactive metabolites, including difficidin, fengycin, and surfactin. Furthermore, a potential class II lanthipeptide biosynthetic gene cluster and genes that are involved in auxin biosynthesis were found. Through the analysis of transcriptome data, we found that the key bacteriostatic genes, as predicted in the genome, exhibited different levels of mRNA expression. Through metabolite isolation, purification, and exposure experiments, we found that a variety of metabolites of WS-8 exert an inhibitory effect on the necrotrophic fungus Botrytis cinerea, which causes gray mold; by mass spectrometry, we found that the main substances are mainly iturins and fengycins. Therefore, this strain has the potential to be utilized as an antifungal agent in agriculture.