Inhibition of LTB4 biosynthesis in situ by CGS 23885, a potent 5-lipoxygenase inhibitor, correlates with its pleural fluid concentrations in an experimentally induced rat pleurisy model

An intrapleural injection of carrageenan in rats induced LTB₄ and LTC₄/D₄/E₄ biosynthesis, exudate formation, and cellular influx in the pleural cavity. An injection of calcium ionophore (A23187, 100nmol) 16–18h after carrageenan injection augmented leukotriene biosynthesis and exudate formation, but not cellular influx. The carrageenan-induced pleurisy model modifid by A23187 administration was used to study the oral effect of CGS 23885 (N-hydroxy-N-[(6-phenoxy-2H-1-benzopyran-3-yl)-methyl]urea), a potent 5-lipoxygenase (5-LO) inhibitor, on inflammatory parameters. CGS 23885 dose-dependently (1 to 30mg/kg) inhibited the enhanced LTB₄ and LTC₄/D₄/E₄ (1 to 10mg/kg) biosynthesis, but had no effect on enhanced exudate formation. An inhibitory effect of CGS 23885 of small magnitude on cellular influx due to carrageenan stimulation was seen at 30mg/kg. The concentrations of CGS 23885 in the pleural fluid were dose-related, and a positive correlation (r ²=0.989) between pleural fluid concentration of LTB₄ and CGS 23885 was observed. The results confirm that CGS 23885 is a specific, orally active 5-LO inhibitor which can achieve concentrations in the pleural cavity sufficient to inhibit production of LTB₄ and LTC₄/D₄/E₄ in an ongoing inflammatory response. Received: 9 February 1995 / Accepted: 20 December 1996     

Fatal intoxication by 5F–ADB and diphenidine: Detection,quantification, and investigation of their main metabolic pathways in humans by LC/MS/MS and LC/Q‐TOFMS

Despite the implementation of a new blanket scheduling system in 2013, new psychoactive substance (NPS) abuse remains a serious social concern in Japan. We present a fatal intoxication case involving 5F–ADB (methyl 2‐[1‐(5‐fluoropentyl)‐1H–indazole‐3‐carboxamido]‐3,3‐dimethylbutanoate) and diphenidine. Postmortem blood screening by liquid chromatography/quadrupole time‐of‐flight mass spectrometry (LC/Q‐TOFMS) in the information‐dependent acquisition mode only detected diphenidine. Further urinary screening using an in‐house database containing NPS and metabolites detected not only diphenidine but also possible 5F–ADB metabolites; subsequent targeted screening by LC/tandem mass spectrometry (LC/MS/MS) allowed for the detection of a very low level of unchanged 5F–ADB in postmortem heart blood. Quantification by standard addition resulted in the postmortem blood concentrations being 0.19 ± 0.04 ng/mL for 5F–ADB and 12 ± 2.6 ng/mL for diphenidine. Investigation of the urinary metabolites revealed pathways involving ester hydrolysis (M1) and oxidative defluorination (M2), and further oxidation to the carboxylic acid (M3) for 5F–ADB. Mono‐ and di‐hydroxylated diphenidine metabolites were also found. The present case demonstrates the importance of urinary metabolite screening for drugs with low blood concentration. Synthetic cannabinoids (SCs) fluorinated at the terminal N‐alkyl position are known to show higher cannabinoid receptor affinity relative to their non‐fluorinated analogues; 5F–ADB is no exception with high CB₁ receptor activity and much greater potency than Δ⁹‐THC and other earlier SCs, thus we suspect its acute toxicity to be high compared to other structurally related SC analogues. The low blood concentration of 5F–ADB may be attributed to enzymatic and/or non‐enzymatic degradation, and further investigation into these possibilities is underway.     

Determination of lansoprazole in human plasma by rapid resolution liquid chromatography–electrospray tandem mass spectrometry: Application to a bioequivalence study on Chinese volunteers

A simple, sensitive and rapid LC/MS/MS method was developed for the quantification of lansoprazole in human plasma. After a simple sample preparation procedure by one-step protein precipitation with acetonitrile, lansoprazole and the internal standard bicalutamide were chromatographed on a Zorbax SB-C₁₈ (3.0 mm × 150 mm, 3.5 μm, Agilent) column with the mobile phase consisted of methanol–water (70:30, v/v, containing 5 mM ammonium formate, pH was adjusted to 7.85 by 1% ammonia solution). Detection was performed on a triple quadrupole tandem mass spectrometry by multiple reaction monitoring (MRM) mode via negative eletrospray ionization source (ESI⁻). The lower limit of quantification was 5.5 ng/mL, and the assay exhibited a linear range of 5.5–2200.0 ng/mL. The validated method was successfully applied to investigate the bioequivalence between two kinds of preparation (test vs. reference product) in twenty-eight healthy male Chinese volunteers.     

Inhibition of the arachidonic acid cascade by norathyriol via blockade of cyclooxygenase and lipoxygenase activity in neutrophils

Recent studies have suggested that dual inhibitors of cyclooxygenase (COX) and lipoxygenase (LO) may be more beneficial in the treatment of inflammatory diseases in which platelet-leukocyte interaction dominates the underlying inflammatory process, than inhibitors of COX or LO alone. In this study, we examined oxygenated xanthones, shown previously to inhibit platelet and neutrophil activation, with respect to the potency of COX inhibition. 1,3,6,7-Tetrahydroxyxanthone (norathyriol) was the most potent. Norathyriol suppressed thromboxane B₂ (TXB₂) and leukotriene B₄ (LTB₄) formation in calcium ionophore (A23187)- and formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated rat neutrophils. Norathyriol was 3–4 times more active against LTB₄ formation than against TXB₂ formation (IC₅₀ about 2.8 vs. 10 µM, respectively). Norathyriol also inhibited prostaglandin D₂ (PGD₂) formation in A23187-stimulated rat mast cells (IC₅₀ 3.0±1.2 µM) and in arachidonic acid (AA)-activated mast cell lysate. Norathyriol was a more effective inhibitor of 5-LO activity than of COX, as shown also by analyses of enzyme activities in a cell-free system, of COX and 5-LO metabolic capacity in neutrophils and of ex vivo TXB₂ and LTB₄ formation in A23187-stimulated neutrophils. Moreover, norathyriol inhibited COX-2 and 12-LO with IC₅₀ values (19.6±1.5 and 1.2±0.1 µM, respectively) similar to those required for the inhibition of COX-1 and 5-LO (16.2±1.5 and 1.8±0.4 µM, respectively). Inhibition of 15-LO by norathyriol was slightly less active. Norathyriol had no effect on A23187-induced AA release from neutrophils and did not affect phospholipase A₂ (PLA₂) activity in a cell-free system. These results indicate that norathyriol inhibits the formation of PGs and LTs in neutrophils probably through direct blockade of COX and 5-LO activities. Norathyriol, a single molecule with multiple targets, might provide a potential therapeutic benefit in the treatment of inflammatory diseases.     

Intracerebral microdialysis coupled to LC–MS/MS for the determination tramadol and its major pharmacologically active metabolite O‐desmethyltramadol in rat brain microdialysates

A rapid and sensitive method involving liquid chromatography electrospray tandem mass spectrometry (LC‐ESI‐MS/MS) coupled to an intracerebral microdialysis technique was developed for the determination and pharmacokinetic investigation of tramadol and its major active metabolite O ‐desmethyltramadol (ODT) in rat brain. The microdialysis samples were separated on a C18 column and eluted with a mobile phase of acetonitrile‐water‐formic acid (50:50:0.1; v/v/v ) at a flow rate of 0.3 mL/min. The ESI‐MS/MS spectra were performed in electrospray positive ion mode, and the analytes were detected by multiple reaction monitoring (MRM) of the transitions m/z [M + H]⁺ 264.3 → 58.2 for tramadol, m/z [M + H]⁺ 250.3 → 58.3 for ODT, and m/z [M + H]⁺ 379.4 → 264.0 for ambroxol (internal standard; IS). The total run time was 4.0 min. A lower limit of quantitation (LLOQ) was achieved as 1 ng/mL for tramadol and 0.5 ng/mL for ODT, with excellent linearity over a concentration range of 1 ~ 500 ng/mL (r  > 0.99) for tramadol and 0.5 ~ 50 ng/mL for ODT (r  > 0.99), respectively. The proposed method was successfully applied to the pharmacokinetic studies of tramadol and ODT in rat brain. Copyright © 2017 John Wiley & Sons, Ltd.     

Acrolein increases 5-lipoxygenase expression in murine macrophages through activation of ERK pathway

Episodic exposure to acrolein-rich pollutants has been linked to acute myocardial infarction, and 5-lipoxygenase (5-LO) is involved in the production of matrix metalloproteinase-9 (MMP-9), which destabilizes atherosclerotic plaques. Thus, the present study determined the effect of acrolein on 5-LO/leukotriene B₄ (LTB₄) production in murine macrophages. Stimulation of J774A.1 cells with acrolein led to increased LTB₄ production in association with increased 5-LO expression. Acrolein-evoked 5-LO expression was blocked by pharmacological inhibition of the ERK pathway, but not by inhibitors for JNK and p38 MAPK pathways. In line with these results, acrolein exclusively increased the phosphorylation of ERK among these MAPK, suggesting a role for the ERK pathway in acrolein-induced 5-LO expression with subsequent production of LTB₄. Among the receptor tyrosine kinases including epidermal growth factor receptor (EGFR) and platelet derived growth factor receptor (PDGFR), acrolein-evoked ERK phosphorylation was attenuated by AG1478, an EGFR inhibitor, but not by AG1295, a PDGFR inhibitor. In addition, acrolein-evoked 5-LO expression was also inhibited by inhibition of EGFR pathway, but not by inhibition of PDGFR pathway. These observations suggest that acrolein has a profound effect on the 5-LO pathway via an EGFR-mediated activation of ERK pathway, leading to acute ischemic syndromes through the generation of LTB₄, subsequent MMP-9 production and plaque rupture.     

Study of the in vitro and in vivo metabolism of the tryptamine 5‐MeO‐MiPT using human liver microsomes and real case samples

The synthetic tryptamine 5‐methoxy‐N‐methyl‐N‐isopropyltryptamine (5‐MeO‐MiPT) has recently been abused as a hallucinogenic drug in Germany and Switzerland. This study presents a case of 5‐MeO‐MiPT intoxication and the structural elucidation of metabolites in pooled human liver microsomes (pHLM), blood, and urine. Microsomal incubation experiments were performed using pHLM to detect and identify in vitro metabolites. In August 2016, the police encountered a naked man, agitated and with aggressive behavior on the street. Blood and urine samples were taken at the hospital and his premises were searched. The obtained blood and urine samples were analyzed for in vivo metabolites of 5‐MeO‐MiPT using liquid chromatography–high resolution tandem mass spectrometry (LC–HRMS/MS). The confiscated pills and powder samples were qualitatively analyzed using Fourier transform infrared (FTIR), gas chromatography–mass spectrometry (GC–MS), LC‐HRMS/MS, and nuclear magnetic resonance (NMR). 5‐MeO‐MiPT was identified in 2 of the seized powder samples. General unknown screening detected cocaine, cocaethylene, methylphenidate, ritalinic acid, and 5‐MeO‐MiPT in urine. Seven different in vitro phase I metabolites of 5‐MeO‐MiPT were identified. In the forensic case samples, 4 phase I metabolites could be identified in blood and 7 in urine. The 5 most abundant metabolites were formed by demethylation and hydroxylation of the parent compound. 5‐MeO‐MiPT concentrations in the blood and urine sample were found to be 160 ng/mL and 3380 ng/mL, respectively. Based on the results of this study we recommend metabolites 5‐methoxy‐N‐isopropyltryptamine (5‐MeO‐NiPT), 5‐hydroxy‐N‐methyl‐N‐isopropyltryptamine (5‐OH‐MiPT), 5‐methoxy‐N‐methyl‐N‐isopropyltryptamine‐N‐oxide (5‐MeO‐MiPT‐N‐oxide), and hydroxy‐5‐methoxy‐N‐methyl‐N‐isopropyltryptamine (OH‐5‐MeO‐MiPT) as biomarkers for the development of new methods for the detection of 5‐MeO‐MiPT consumption, as they have been present in both blood and urine samples.     

Metabolism and pharmacokinetics in rats of ganoderiol F,a highly cytotoxic and antitumor triterpene from <Emphasis Type="Italic">Ganoderma lucidum</Emphasis>

The metabolism of ganoderiol F (GF), a cytotoxic and antitumor triterpene from Ganoderma lucidum, by intestinal bacteria and its pharmacokinetics in rats were investigated by using liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). GF was converted to ganodermatriol by anaerobic incubation with bacterial mixtures from rats and humans. This metabolite was detected in rat feces, but not in plasma and urine, after oral administration of GF. The fate of GF after oral (p.o.) and intravenous (i.v.) administration to rats was examined in pharmacokinetics studies. Plasma samples pretreated by solid-phase extraction were quantified by HPLC/MS/MS over a GF concentration range of 1.25–100 ng/ml (S/N = 5). The intra- and interday precision (CV%) was below 8% and accuracy was within the range of 95.9–103.6% for all samples. The range of recovery ratios was 89.2–98.2%. After the administration of GF at 0.5 mg/kg i.v., the plasma concentrations of GF quickly declined and the elimination half-life values (t _1/2α and t _1/2β) were about 2.4 and 34.8 min. On the other hand, the elimination half-life values (t _1/2α) after p.o. administration of GF at doses of 20 and 50 mg/kg were 14.4 and 143.3 min for the former, and 18.6 and 114.6 min for the latter. The AUC_0–t value was 11.17 (ng/ml) h at a GF dose of 0.5 mg/kg i.v., but 49.4 and 111.6 (ng/ml) h at GF doses of 20 and 50 mg/kg p.o., respectively, indicating that the AUC_0–t value is proportional to the administered oral doses. The estimated absolute bioavailability of GF in rats was F = 0.105.     

A direct LC/MS/MS method for the determination of ciclopirox penetration across human nail plate in in vitro penetration studies

Due to severe chelating effect caused by N-hydroxylpyridone group of ciclopirox, there is no published direct HPLC or LC/MS/MS method for the determination of ciclopirox in any in vitro or in vivo matrix. Instead, the time-consuming pre-column derivatization methods have been adapted for indirect analysis of ciclopirox. After overcoming the chelating problem by using K₂EDTA coated tubes, a direct, sensitive and high-throughput LC/MS/MS method was successfully developed and validated to determine the amount of ciclopirox that penetrated across the nail plate during in vitro nail penetration studies. The method involved adding a chemical analog, chloridazon as internal standard (IS) in K₂EDTA coated tubes, mixing IS with ciclopirox in a 96-well plate and then proceeding to LC/MS/MS analysis. The MS/MS was selected to monitor m/z 208.0 → 135.8 and 221.8 → 77.0 for ciclopirox and IS, respectively, using positive electrospray ionization. The method was validated over a concentration range of 8–256 ng/mL, yielding calibration curves with correlation coefficients greater than 0.9991 with a lower limit of quantitation (LLOQ) of 8 ng/mL. The assay precision and accuracy were evaluated using quality control (QC) samples at three concentration levels. Analyzed concentrations ranged from 101% to 113% of their respective nominal concentration levels with coefficients of variation (CV) below 10.6%. The average recovery of ciclopirox from nail matrix was 101%.     

Inhibition of Leukotriene Biosynthesis and Polymorphonuclear Leukocyte Functions by Orally Active Quinolylmethoxyphenylamines

Abstract: The N-substituted quinolylmethoxyphenylamines, ETH603, ETH615 and ETH647, inhibited the formation of LTB₄ in rat peritoneal leukocytes, human peripheral polymorphonuclear leukocytes and canine whole blood. In rat and human cells, the compounds also inhibited the formation of 5-HETE and stimulated the synthesis of 15-HETE. In rat leukocytes, the compounds were 15–30 times more potent inhibitors of LTB₄ synthesis than nordihydroguaiaretic acid, but in canine whole blood they were significantly less potent, possibly due to protein binding. However, after oral administration of the compounds to dogs a long-lasting inhibition of LTB₄ production in peripheral blood was observed at serum concentrations much lower than those required in vitro. Furthermore, the compounds inhibited the LTB₄-directed chemotaxis and the phagocytosis of C. albicans blastospores by canine polymorphonuclear leukocytes both in vitro and following oral administration. The calcium ionophore A23187-induced release of LTB₄ in the peritoneal cavity of rats was also inhibited by systemic administration of the compounds. We therefore conclude that these novel quinolines are orally active 5-lipoxygenase inhibitors which may accumulate in inflammatory cells in vivo, leading to potent inhibition of leukotriene biosynthesis and cell function.     

HPLC–MS/MS method for the intracellular determination of ribavirin monophosphate and ribavirin triphosphate in CEMss cells

A sensitive and specific method using high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) for the determination of ribavirin monophosphate (RBV-MP) and ribavirin triphosphate (RBV-TP) in cells has been developed and validated. In this method, ribavirin phosphorylated metabolites were extracted and separated by anion exchange solid phase extraction (SPE). The RBV-MP and RBV-TP fractions were dephosphorylated using acid phosphatase and further purified by phenyl boronate SPE prior to HPLC–MS/MS analysis. ¹³C₅-uridine was added as internal standard to obtain better accuracy and precision of the analysis. The MS/MS detector was optimized at multiple reaction monitoring (MRM) using positive electrospray ionization to detect 245 → 113 and 250 → 133 transitions for ribavirin and internal standard, respectively. The calibration curve was linear over a concentration range of 0.01–10 μg/mL with a limit of quantitation of 0.01 μg/mL. Mean inter-assay accuracy and precision for RBV-MP and RBV-TP quality control samples at 0.03, 0.3 and 8 μg/mL were 5% and 10%, respectively. This method was successfully used for the in vitro determination of RBV-MP and RBV-TP in CEM_ss cells cultured with RBV.     

Development and validation of a high-performance liquid chromatography/tandem mass spectrometry method for the determination of artemether and its active metabolite dihydroartemisinin in human plasma

To study the pharmacokinetic profile of artemether in children and in the context of antiviral drugs for HIV infected patients co-infected with malaria, an LC-MS/MS method was developed and validated to simultaneously determine artemether and its metabolite dihydroartemisinin in human plasma. Using artemisinin as the internal standard, 0.5 mL samples were processed with solid phase extraction (Waters Oasis^® HLB column), the elutes were directly injected onto a C₁₈ LC column (Waters, Symmetry^®, 150 mm × 4.6 mm, 5 μm). Mass detection utilized ESI⁺ as the ionization mode and MRM as the quantitation mode. In respect to the low ionization capacity of artemether, ammonium formate was added to the LC mobile phase to facilitate ionization (M+NH₄⁺). The calibration range was 2–200 ng/mL. The recovery was 73–81% for artemether and 90–99% for dihydroartemisinin. The validated method was applied to analysis of clinical samples with results in good agreement with an existing method.     

LC-MS/MS method for determination of hederacolchiside E, a neuroactive saponin from Pulsatilla koreana extract in rat plasma for pharmacokinetic study

A simple, rapid, and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was applied to pharmacokinetic study of a neuroactive oleanolic-glycoside saponin, hederacolchiside E from SK-PC-B70M, a standardized extract of Pulsatilla koreana in rat. Rat plasma samples were pretreated by protein precipitation with acetonitrile, eluted from C₁₈ column, and analyzed using electrospray ionization (ESI)-MS/MS in negative ion mode. Digoxin was used as an internal standard. The standard curves were linear (r > 0.997) over the concentration ranges of 2–500 ng/mL. The intra- and inter-day precisions were measured to be below 9% and accuracy between 90 and 111% for all quality control samples at 2, 20, 100, and 500 ng/mL (n = 5). The lower limits of quantification (LLOQ) for hederacolchiside E was 2 ng/mL and the limit of detection (LOD) 0.5 ng/mL using 20 μL of plasma sample. Subsequently, hederacolchiside E was determined in rat plasma samples after oral administration of SK-PC-B70M. The mean maximum plasma concentrations of hederacolchiside E were 0.07, 0.13, and 0.36 μg/mL and the mean areas under the plasma concentration versus time curve 0.56, 1.27, and 6.46 μg h/mL at doses of 100, 200, and 400 mg/kg, respectively, which indicated non-linear pharmacokinetic pattern. In conclusion, this method was successfully applied to the pharmacokinetic study of hederacolchiside E after an oral administration of SK-PC-B70M to rats.     

Determination of tegafur, 5-fluorouracil,gimeracil and oxonic acid in human plasma using liquid chromatography–tandem mass spectrometry

S-1 is an oral anticancer drug, which consists of tegafur (FT), gimeracil (CDHP) and potassium oxonate (Oxo) at a molar ratio of 1:0.4:1. Among these, tegafur is a prodrug, and is rapidly metabolized to the active drug, 5-fluorouracil (5-FU), in vivo. To evaluate the pharmacokinetics of S-1 in patients, LC–MS/MS methods were developed and validated for determination of FT, 5-FU, CDHP and Oxo in human plasma. FT, 5-FU and CDHP were extracted from plasma following protein precipitation, separated on a Synergi Hydro-RP column and simultaneously quantified by LC–MS/MS. The mobile phase consisted of methanol–water–ammonia–acetic acid (27:73:0.0018:0.018, v/v/v/v). The mass spectrometer was operated in negative mode using electrospray ionization. The calibration curves were linear in the range of 12.0–3000 ng/mL for FT, and 2.00–500 ng/mL for 5-FU and CDHP. The accuracy ranged from 93.1% to 110.7% and the precision ranged from 2.4% to 14.6% for each analyte. To determine Oxo in human plasma, an LC–MS/MS method employing pre-column derivatization was developed and validated. 4-Bromomethyl-7-methoxycoumarin was chosen as the derivatization reagent and [¹³C₂,¹⁵N₃]-Oxo was used as the internal standard. The MS/MS detection was operated in positive mode using an APCI source. The calibration range was 2.00–150 ng/mL. The accuracy and precision were within 95.9–99.1% and 4.4–10.0%, respectively. The validated methods were successfully applied to characterize the pharmacokinetic profiles of FT, 5-FU, CDHP and Oxo following oral administration of 60 mg S-1 tablets to patients with solid gastrointestinal tract tumors.     

The risk of higenamine adverse analytical findings following oral administration of plumula nelumbinis capsules

Higenamine is a β2‐agonist that has been included in the Prohibited List of the World Anti‐Doping Agency (WADA) since 2017. Meanwhile, it exists in plumula nelumbinis, a part of the lotus seed, and is commonly used as an ingredient in cuisines, herbal medicines, and nutritional supplements in China and other countries in East Asia. Therefore, an evaluation of the risk of an adverse analytical finding (AAF) of higenamine caused by plumula nelumbinis products is necessary in doping control. In this study, 14 volunteers took plumula nelumbinis capsules orally (0.34 g/caplet, 6 caplets/day, 7 days), and another 11 volunteers ingested higenamine tablets (three 5 mg tablets/day for 7 days). Urine samples were collected over a period of 14 days. All urine samples were subjected to quantitative dilute‐and‐shoot analysis using liquid chromatography–tandem mass spectrometry (LC–MS/MS). The analytical results showed that urinary higenamine concentrations exceeded the WADA reporting limit (10 ng/mL) during the drug period in most sample groups. The maximum higenamine concentration observed in the plumula nelumbinis capsule group was 500 ng/mL. Based on confidence interval theory, appropriate data were used to establish mathematical models. The models reflected that the higenamine concentration in urine can exceed the WADA reporting limit with a high probability after taking plumula nelumbinis capsules. In conclusion, oral administration of plumula nelumbinis capsules showed a high risk of an AAF due to higenamine.     

4-Hydroxynonenal enhances MMP-9 production in murine macrophages via 5-lipoxygenase-mediated activation of ERK and p38 MAPK

Exaggerated levels of 4-hydroxynonenal (HNE) and 5-lipoxygenase (5-LO) co-exist in macrophages in atherosclerotic lesions, and activated macrophages produce MMP-9 that degrades atherosclerotic plaque constituents. This study investigated the effects of HNE on MMP-9 production, and the potential role for 5-LO derivatives in MMP-9 production in murine macrophages. Stimulation of J774A.1 cells with HNE led to activation of 5-LO, as measured by leukotriene B₄ (LTB₄) production. This was associated with an increased production of MMP-9, which was blunted by inhibition of 5-LO with MK886, a 5-LO inhibitor or with 5-LO siRNA. A cysteinyl-LT₁ (cysLT₁) receptor antagonist, REV-5901 as well as a BLT₁ receptor antagonist, U-75302, also attenuated MMP-9 production induced by HNE. Furthermore, LTB₄ and cysLT (LTC₄ and LTD₄) enhanced MMP-9 production in macrophages, suggesting a pivotal role for 5-LO in HNE-mediated production of MMP-9. Among the MAPK pathways, LTB₄ and cysLT enhanced phosphorylation of ERK and p38 MAPK, but not JNK. Linked to these results, a p38 MAPK inhibitor as well as an ERK inhibitor blunted MMP-9 production induced by LT. Collectively, these data suggest that 5-LO-derived LT mediates HNE-induced MMP-9 production via activation of ERK and p38 MAPK pathways, consequently leading to plaque instability in atherosclerosis.     

Rapid simultaneous analysis of cyclooxygenase, lipoxygenase and cytochrome P-450 metabolites of arachidonic and linoleic acids using high performance liquid chromatography/mass spectrometry in tandem mode

Eicosanoids are oxidized arachidonate-derived lipid products generated by cyclooxygenase, lipoxygenase and cytochrome P-450 pathways. They are involved in diverse processes in health and disease and they are highly bioactive. Gas chromatography and enzyme immunoassays were used to quantify these mediators in the past. However, the recent availability of high-sensitivity liquid chromatography-mass spectrometry has provided a new approach for quantification that minimizes the sample size and the required preparation. This paper describes a rapid and simple technique for the simultaneous quantitative analysis of prostaglandin (PG) E₂ and PGJ₂; leukotrienes (LT) B₄ and D₄; 5-, 12-, 15- and 20-hydroxyeicosatetraenoic acids (HETEs); 13-hydroxyoctadecadienoic acid (13-HODE); 5,6-, 8,9-, 11,12- and 14,15-epoxyeicosatrienoic acids (EETs); and 11,12- and 14,15-dihydroxieicosatrienoic acids (DHETs) in cell culture supernatants and urine. We simultaneously analyzed 14 arachidonic acid metabolites representative from the three pathways, together with 13-HODE, a linoleic-derived product. Solid phase extraction was used for the sample preparation. The recoveries obtained ranged from 25% to 100%, depending on the metabolites. The LC/MS/MS method used the gradient on a C₁₈ column and electrospray ionization in negative ion detection mode. The method was optimized for sensitivity and for separation within 20 min. The linear ranges of the calibration curves were 0.1-200 ng/ml for PGE₂, PGJ₂, LTB₄, 5-HETE, 12-HETE, 15-HETE, 13-HODE, 11,12-EET, 11,12-DHET and 14,15-DHET, and 1-200 ng/ml for LTD₄, 20-HETE, 5,6-EET, 8,9-EET and 14,15-EET. The advantages of this method include minimal sample preparation, high sensitivity and elimination of the problem associated with thermal instability in gas chromatography analysis.     

Analytical quantification,intoxication case series,and pharmacological mechanism of action for N‐ethylnorpentylone (N‐ethylpentylone or ephylone)

Synthetic cathinones continue to proliferate in clandestine drug markets worldwide. N‐ethylnorpentylone (also known as N‐ethylpentylone or ephylone) is a popular emergent cathinone, yet little information is available about its toxicology and pharmacology. Here we characterize the analytical quantification, clinical presentation, and pharmacological mechanism of action for N‐ethylnorpentylone. Liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) was used to quantify N‐ethylnorpentylone in blood obtained from human cases. Clinical features exhibited by the intoxicated individuals are described. The activity of N‐ethylnorpentylone at plasma membrane transporters for dopamine (DAT), norepinephrine (NET) and 5‐HT (SERT) was assessed using in vitro assays measuring uptake inhibition and evoked release of [³H] neurotransmitters in rat brain synaptosomes. Our LC–MS/MS method assayed N‐ethylnorpentylone concentrations with limits of detection and quantification of 1 and 5 ng/mL, respectively. Quantitation was linear from 5 to 500 ng/mL, and the method displayed specificity and reproducibility. Circulating concentrations of N‐ethylnorpentylone ranged from 7 to 170 ng/mL in clinical cases, and the associated symptoms included palpitations, tachycardia, agitation, hallucinations, coma and death. N‐Ethylnorpentylone was a potent inhibitor at DAT (IC₅₀ = 37 nM), NET (IC₅₀ = 105 nM) and SERT (IC₅₀ = 383 nM) but displayed no transporter releasing activity. We present a validated method for quantifying N‐ethylnorpentylone in human case work. The drug is a psychomotor stimulant capable of inducing serious cardiovascular and neurological side‐effects which can be fatal. In vitro findings indicate that N‐ethylnorpentylone exerts its effects by potent blockade of DAT and NET, thereby elevating extracellular levels of dopamine and norepinephrine in the brain and periphery.     

Leukotriene B4 receptor antagonists as therapeutics for inflammatory disease: preclinical and clinical developments

Leukotriene B₄ (LTB₄) is a lipid inflammatory mediator derived from membrane phospholipids by the sequential actions of cytosolic phospholipase A2 (PLA2), 5-lipoxygenase (5-LO) and leukotriene A₄ (LTA₄) hydrolase. Several inflammatory diseases, including asthma, chronic obstructive pulmonary disease, arthritis and inflammatory bowel disease, have been associated with elevated levels of LTB₄. As a result, pharmacological strategies to modulate the synthesis of LTB₄ (inhibition of PLA2, 5-LO or LTA₄ hydrolase) or the effects of LTB₄ itself (antagonism of LTB₄ receptors) are being developed by several companies. Two G-protein-coupled receptors mediate the effects of LTB₄, namely BLT1 and BLT2. The pharmacology, expression and function of these two receptors were last reviewed by Tager and Luster in 2004. Since then, there has been an increased understanding of the function of these receptors, in particular for the lesser understood of the two receptors, BLT2. Furthermore, since last reviewed in 1996, there have been several clinical developments in the use of BLT receptor antagonists for inflammatory diseases. This review summarizes the latest preclinical and clinical developments in BLT antagonism for inflammatory diseases and discusses potential future developments.     

Development and validation of a liquid chromatography–tandem mass spectrometry assay for the simultaneous quantitation of prednisolone and dipyridamole in human plasma and its application in a pharmacokinetic study

We have developed and validated an accurate, sensitive, and robust LC–MS/MS method that determines the concentration of CRx-102 (the combination of prednisolone and dipyridamole) in human plasma. In this method, prednisolone, dipyridamole, and the combined internal standards (IS) prednisolone-d₆ (IS for prednisolone) and dipyridamole-d₂₀ (IS for dipyridamole) were extracted from 100 μL human EDTA plasma using methylbutyl ether. Calibration curves were linear over a concentration range of 0.4–200 ng/mL for prednisolone and 5–3000 ng/mL for dipyridamole. The analytes were quantitatively determined using tandem mass spectrometry operated in positive electrospray ionization in a multiple reaction monitoring (MRM) mode. This validated method has been used successfully in clinical pharmacokinetic studies of CRx-102 in healthy volunteers.