Investigation of the pharmacokinetics and metabolic fate of Fasiglifam (TAK-875) in male and female rats following oral and intravenous administration.

The metabolism and pharmacokinetics of fasiglifam (TAK-875, 2-[(3S)-6-[[3-[2,6-dimethyl-4-(3-methylsulfonylpropoxy)phenyl]phenyl]methoxy]-2,3-dihydro-1-benzofuran-3-yl]acetic acid), a selective free fatty acid receptor 1 (FFAR1)/GPR40 agonist, were studied following intravenous (5 mg/kg) and oral administration (10 and 50 mg/kg) to male and female Sprague Dawley rats.Following intravenous dosing at 5 mg/kg, peak observed plasma concentrations of 8.8/9.2 µg/ml were seen in male and female rats respectively.Following oral dosing, peak plasma concentrations at 1 h of ca. 12.4/12.9 µg/ml for 10 mg/kg and 76.2/83.7 µg/ml for 50 mg/kg doses were obtained for male and female rats respectively. Drug concentrations then declined in the plasma of both sexes with t1/2's of 12.4 (male) and 11.2 h (female). Oral bioavailability was estimated to be 85-120% in males and females at both dose levels.Urinary excretion was low, but in a significant sex-related difference, female rats eliminated ca. 10-fold more drug-related material by this route.Fasiglifam was the principal drug-related compound in plasma, with 15 metabolites, including the acyl glucuronide, also detected. In addition to previously identified metabolites, a novel biotransformation, that produced a side-chain shortened metabolite via elimination of CH2 from the acetyl side chain was noted with implications for drug toxicity.

Enhanced chromatographic efficiency obtained with vacuum jacketed columns facilitates the rapid UHPLC/MS/MS-based analysis of fasiglifam in rat plasma.

The use of vacuum jacketed LC columns (VJC) to minimize on- and post-column band broadening to maximize chromatographic performance has been evaluated as a potential route to improved high throughput (HT) analysis. Here the use of the "VJC" approach has been applied to the HT bioanalysis of the antidiabetic GPR40 agonist drug fasiglifam in rat plasma samples obtained following a 5 mg/kg IV dose. The data obtained from a 1 minute VJC/MS-based analysis showed significant improvements compared to that from a conventional 2 minute UHPLC method for the drug. Notably, using VJC/MS with the rapid 1 min analysis provided a ca. 50% reduction in peak width coupled with a 2-5 fold higher peak response whilst doubling analytical throughput when compared to a conventional UHPLC/MS method. In addition, the increased resolution provided by the VJC system also improved the separation of fasiglifam from common matrix interferences such as co-extracted phospholipids thereby reducing the potential for matrix effects. The concatenation of these improvements suggests that the VJC approach may indeed provide a pathway to more sensitive, robust and high throughput drug bioanalysis, with particular advantages for drug discovery applications.

Plasma fetal bile acids 7α-hydroxy-3-oxochol-4-en-24-oic acid and 3-oxachola-4,6-dien-24-oic acid indicate severity of liver cirrhosis.

Two 3-oxo-Δ4 fetal bile acids, 3-oxachola-4,6-dien-24-oic acid (1) and 7α-hydroxy-3-oxochol-4-en-24-oic acid (2), occur normally in the human fetus but remain elevated in neonates and children with severe cholestatic liver disease due to an autosomal recessive inborn error of metabolism affecting Δ4-3-oxo-steroid 5ß-reductase (AKR1D1). Relatively little is known about 1 and 2 in adult patients with liver disease. The chemical synthesis of 1 and 2 is therefore described and their quantitation in plasma by ultrarapid chromatography-triple quadrupole mass spectrometry. Plasma concentrations of 1 and 2 were investigated in 25 adult patients with varying degrees of liver cirrhosis with and without hepatocellular carcinoma (HCC). Highly statistically significant correlations (P < 0.0001) were found between severity of liver cirrhosis, determined by the Child-Pugh and MELD scores, with plasma 1 and 2 concentrations, both alone and combined. The presence of HCC did not influence these correlations. Plasma cholic, chenodeoxycholic, deoxycholic, lithocholic or ursodeoxycholic acids, free and as their glycine or taurine conjugates, did not correlate with Child-Pugh or MELD score when corrected for multiple comparisons. These findings demonstrate that plasma levels of fetal bile acids 3-oxachola-4,6-dien-24-oic acid and 7α-hydroxy-3-oxochol-4-en-24-oic acid and likely deteriorating AKR1D1 activity indicate the severity of liver cirrhosis measured by the Child-Pugh and MELD scores.

Rapid determination of the pharmacokinetics and metabolic fate of gefitinib in the mouse using a combination of UPLC/MS/MS, UPLC/QToF/MS, and ion mobility (IM)-enabled UPLC/QToF/MS.

The metabolism and pharmacokinetics of gefitinib (Iressa®, N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholino-propoxy)quinazolin-4-amine), a selective thymidylate kinase inhibitor for the epidermal growth factor receptor (EGFR), was studied after IV and PO administration to male C57BL6 mice at 10 and 50 mg/kg respectively.The pharmacokinetics and metabolism of gefitinib were investigated using a range of rapid UHPLC-MS and UHPLC-IM-HRMS methods, using both reversed-phase (RP) and hydrophilic interaction liquid chromatography (HILIC), to rapidly determine the drugs pharmacokinetics and metabolic fate.Rapid oral absorption resulted in peak plasma concentrations at 1 h of ca. 7 µg/mL, that declined with a half-life of 3.8 h (2.6 h for the IV route), and providing an estimated oral bioavailability of 53%. Gefitinib itself was the major circulating drug-related compound in plasma extracts, with a total of 11 metabolites identified.The urinary profiles determined using both HILIC and RP-UPLC-IM-MS detected gefitinib and 10 metabolites or 15 metabolites respectively including the detection of a number of novel glucuronide conjugates.Despite rapid, sub 5 min, LC profiling methods being employed metabolite coverage was shown to be high and compared well with that of previous studies.

Evaluation of cyclooxygenase oxylipins as potential biomarker for obesity-associated adipose tissue inflammation and type 2 diabetes using targeted multiple reaction monitoring mass spectrometry.

INTRODUCTION: Obesity is associated with adipose tissue inflammation which in turn drives insulin resistance and the development of type 2 diabetes. Oxylipins are a collection of lipid metabolites, subdivided in different classes, which are involved in inflammatory cascades. They play important roles in regulating adipose tissue homeostasis and inflammation and are therefore putative biomarkers for obesity-associated adipose tissue inflammation and the subsequent risk of type 2 diabetes onset. The objective for this study is to design an assay for a specific oxylipin class and evaluate these as potential prognostic biomarker for obesity-associated adipose tissue inflammation and type 2 diabetes. METHODS: An optimized workflow was developed to extract oxylipins from plasma using solid-phase extraction followed by analysis using ultra-high performance liquid chromatography coupled to a triple quadrupole mass spectrometer in multiple reaction monitoring mode. This workflow was applied to clinical plasma samples obtained from obese-type 2 diabetes patients and from lean and obese control subjects. RESULTS: The assay was analytically validated and enabled reproducible analyses of oxylipins extracted from plasma with acceptable sensitivities. Analysis of clinical samples revealed discriminative values for four oxylipins between the type 2 diabetes patients and the lean and obese control subjects, viz. PGF2α, PGE2, 15-keto-PGE2 and 13,14-dihydro-15-keto-PGE2. The combination of PGF2α and 15-keto-PGE2 had the most predictive value to discriminate type 2 diabetic patients from lean and obese controls. CONCLUSIONS: This proof-of-principle study demonstrates the potential value of oxylipins as biomarkers to discriminate obese individuals from obese-type 2 diabetes patients.

Advances in quadrupole and time-of-flight mass spectrometry for peptide MRM based translational research analysis.

The application of unit resolution tandem quadrupole and high-resolution orthogonal acceleration ToF mass spectrometers for the quantitation and translational analysis of proteolytic peptides is described. The MS platforms were contrasted in terms of sensitivity and linear response. Moreover, the selectivity of the platforms was investigated and the effect on quantitative precision studied. Chromatographic LC conditions, including gradient length and configuration, were investigated with respect to speed/throughput, while minimizing isobaric interferences, thereby providing information with regard to practical sample cohort size limitations of LC-MS for large cohort experiments. In addition to these fundamental analytical performance metrics, precision and linear dynamic ranges were also studied. An LC-MS configuration that encompasses the best combination of throughput and analytical accuracy for translational studies was chosen, despite the MS platforms giving similar quantitative performance, and instances were identified where alternative combinations were found to be beneficial. This configuration was utilized to demonstrate that proteolytically digested nondepleted samples from heart failure patients could be classified with good discriminative power using a subset of proteins previously suggested as candidate biomarkers for cardiovascular diseases.