Repurposing of Omarigliptin as a Neuroprotective Agent Based on Docking with A2A Adenosine and AChE Receptors, Brain GLP-1 Response and Its Brain/Plasma Concentration Ratio after 28 Days Multiple Doses in Rats Using LC-MS/MS.

The authors in the current work suggested the potential repurposing of omarigliptin (OMR) for neurodegenerative diseases based on three new findings that support the preliminary finding of crossing BBB after a single dose study in the literature. The first finding is the positive results of the docking study with the crystal structures of A2A adenosine (A2AAR) and acetylcholine esterase (AChE) receptors. A2AAR is a member of non-dopaminergic GPCR superfamily receptor proteins and has essential role in regulation of glutamate and dopamine release in Parkinson's disease while AChE plays a major role in Alzheimer's disease as the primary enzyme responsible for the hydrolytic metabolism of the neurotransmitter acetylcholine into choline and acetate. Docking showed that OMR perfectly fits into A2AAR binding pocket forming a distinctive hydrogen bond with Threonine 256. Besides other non-polar interactions inside the pocket suggesting the future of the marketed anti-diabetic drug (that cross BBB) as a potential antiparkinsonian agent while OMR showed perfect fit inside AChE receptor binding site smoothly because of its optimum length and the two fluorine atoms that enables quite lean fitting. Moreover, a computational comparative study of OMR docking, other 12 DPP-4 inhibitors and 11 SGLT-2 inhibitors was carried out. Secondly, glucagon-like peptide-1 (GLP-1) concentration in rats' brain tissue was determined by the authors using sandwich GLP-1 ELISA kit bio-analysis to ensure the effect of OMR after the multiple doses' study. Brain GLP-1 concentration was elevated by 1.9-fold following oral multiple doses of OMR (5 mg/kg/day, p.o. for 28 days) as compared to the control group. The third finding is the enhanced BBB crossing of OMR after 28 days of multiple doses that had been studied using LC-MS/MS method with enhanced liquid-liquid extraction. A modified LC-MS/MS method was established for bioassay of OMR in rats' plasma (10-3100 ng/mL) and rats' brain tissue (15-2900 ng/mL) using liquid-liquid extraction. Alogliptin (ALP) was chosen as an internal standard (IS) due to its LogP value of 1.1, which is very close to the LogP of OMR. Extraction of OMR from samples of both rats' plasma and rats' brain tissue was effectively achieved with ethyl acetate as the extracting solvent after adding 1N sodium carbonate to enhance the drug migration, while choosing acetonitrile to be the diluent solvent for the IS to effectively decrease any emulsion between the layers in the stated method of extraction. Validation results were all pleasing including good stability studies with bias of value below 20%. Concentration of OMR in rats' plasma were determined after 2 h of the latest dose from 28 days multiple doses, p.o, 5 mg/kg/day. It was found to be 1295.66 ± 684.63 ng/mL estimated from the bio-analysis regression equation. OMR passed through the BBB following oral administration and exhibited concentration of 543.56 ± 344.15 ng/g in brain tissue, taking in consideration the dilution factor of 10. The brain/plasma concentration ratio of 0.42 (543.56/1295.66) was used to illustrate the penetration power through the BBB after the multiple doses for 28 days. Results showed that OMR passed through the BBB more effectively in the multiple dose study as compared to the previously published single dose study by the authors. Thus, the present study suggests potential repositioning of OMR as antiparkinsonian agent that will be of interest for researchers interested in neurodegenerative diseases.

Analysis of five active ingredients of Er-Zhi-Wan, a traditional Chinese medicine water-honeyed pill, using the biopharmaceutics classification system.

Er-Zhi-Wan (EZW) is a traditional Chinese medicine with many clinical applications and used as a health product in East Asia. Five active ingredients (salidroside, specnuezhenide, nuezhenoside, luteolin, and oleanolic acid) were screened out from EZW to develop an in vitro rapid evaluation method for the classification of in vivo drug absorption behavior by biopharmaceutics classification system (BCS). Ultra-performance liquid chromatography was used for quantitative analysis. Solubility and permeability were assayed by equilibrium solubility and multiple models: everted rat intestinal sac model, cultured Caco-2 cells, octanol-water partition coefficient (LogP) method. The BCS properties of drugs were predicted using software applications, and the correlations of measured and predicted values of factors affecting oral drug absorption were calculated. The results were verified by measuring the absolute bioavailability of the active ingredients. Salidroside, specnuezhenide, and nuezhenoside were classified as BCS class III drugs, and luteolin was classified as a BCS class III/I drug because of the difference in LogP and intestinal permeability. Oleanolic acid was classified as a BCS class II/IV drug in acidic media and BCS class I/III drug in other media. Overall, EZW may be classified as a BCS class III drug, and permeability was identified as the primary factor limiting absorption. The results provide a novel method for the evaluation of the in vivo absorption of oral traditional Chinese medicines.

Enhanced Extraction Technique of Omarigliptin from Human Plasma-Applied to Biological Samples from Healthy Human Volunteers.

Enhancing drug extraction from human plasma is a challenging approach that critically affects pharmacokinetic and any further clinical studies based on the drug Cmin and Cmax values. It also has a serious impact on the sensitivity and the lower limit of quantification (LLOQ) value of the bio-analytical methods. An advanced liquid chromatography tandem mass spectrometry (LC-MS/MS) bio-analytical method of omarigliptin (25-1000 nM) was established in human plasma using one-step liquid-liquid extraction. Alogliptin was used as an internal standard (IS) to attain good recovery and reproducibility while reducing the effects of the matrix. Enhanced plasma extraction of omarigliptin was successfully achieved with tertiary butyl methyl ether-diethyl ether (TBME-DEE) mixture as the extracting solvent, while using acetonitrile as the diluent solvent for the IS to effectively decrease the formed emulsion. Multiple Reaction Monitoring (MRM) of the transition pairs of m/z 399.2 to 153.0 for omarigliptin and m/z 340.2 to 116.0 for alogliptin was employed in positive Electro Spray Ionization (ESI) mode. Human plasma samples were collected after 1.5 h (tmax) of Marizev® (12.5 mg) tablets administration to healthy human volunteers showing average concentration of 292.18 nM. Validation results were all satisfactory including successful stability studies with bias below 12%. The proposed study will be valuable for ethnicity comparison studies that will be commenced on omarigliptin in Egypt by the authors in prospective study, following the FDA recommends, to evaluate possible sub-group dissimilarities that include pharmacokinetic parameters.

Effects of D-α-tocopherol polyethylene glycol succinate-emulsified poly(lactic-co-glycolic acid) nanoparticles on the absorption, pharmacokinetics, and pharmacodynamics of salinomycin sodium.

Although salinomycin sodium (SS) has shown in-vitro potential to inhibit cancer stem cell growth and development, its low water solubility makes it a poor candidate as an oral chemotherapeutic agent. To improve the bioavailability of SS, SS was encapsulated here using D-α-tocopherol polyethylene glycol succinate (TPGS)-emulsified poly(lactic-co-glycolic acid) (PLGA) nanoparticles and compared with its parent SS in terms of absorption, pharmacokinetics, and efficacy in suppressing nasopharyngeal carcinomas stem cells. The pharmacokinetics of SS and salinomycin sodium-loaded D-α-tocopherol polyethylene glycol succinate-emulsified poly(lactic-co-glycolic acid) nanoparticles (SLN) prepared by nanoprecipitation were analyzed in-vivo by timed-interval blood sampling and oral administration of SS and SLN to rats. Sensitive liquid chromatography-mass spectrometry (LC-MS) was developed to quantify plasma drug concentrations. SS and SLN transport in Caco-2 cells was also investigated. The therapeutic efficacy of SS and SLN against cancer stem cells was determined by orally administering the drugs to mice bearing CNE1 and CNE2 nasopharyngeal carcinoma xenografts and then evaluating CD133 cell proportions and tumorsphere formation. The in-vivo trial with rats showed that the Cmax, AUC(0-t), and Tmax for orally administered SLN were all significantly higher than those for SS (P<0.05). These findings were corroborated by a Caco-2 cell Transwell assay showing that relative SLN absorption was greater than that of SS on the basis of their apparent permeability coefficients (Papp). Significantly, therapeutic SLN efficacy against nasopharyngeal carcinoma stem cells was superior to that of SS. TPGS-emulsified PLGA nanoparticles effectively increase SS solubility and bioavailability. SLN is, therefore, promising as an oral chemotherapeutic agent against cancer stem cells.

Pharmacokinetic-pharmacodynamic (dipeptidyl peptidase-4 inhibition) model to support dose rationale in diabetes patients, including those with renal impairment, for once-weekly administered omarigliptin.

AIMS: To characterize the population pharmacokinetics (PK) and pharmacodynamics (PD) of the once-weekly dipeptidyl peptidase-4 (DPP-4) inhibitor omarigliptin in healthy subjects and patients with type 2 diabetes mellitus, and use these models to support the dosing recommendation for patient labelling including patients with renal impairment. METHODS: PK and PD were assessed from a total of 9827 omarigliptin concentrations collected from 1387 healthy subjects and patients participating in Phase 1, 2 and 3 studies examining single- or multiple-dose weekly administration of omarigliptin at doses ranging from 0.25 to 400 mg. Population PK and PD analyses were performed using nonlinear mixed effect modelling. RESULTS: A semi-mechanistic 2-compartment model with linear unbound clearance and concentration-dependent binding of omarigliptin to the DPP-4 enzyme in both the central and peripheral compartments adequately described omarigliptin PK. Key covariates on omarigliptin PK included reduced unbound clearance with renal impairment. A direct effect sigmoid maximum inhibitory efficacy model adequately described the relationship between omarigliptin plasma concentrations and DPP-4 inhibition. These models supported the current Japan label instructions that the approved omarigliptin 25-mg once-weekly dose be halved in patients with severe renal impairment and in those with end-stage renal disease. Also, if patients missed a dose, the next dose of omarigliptin should be taken as soon as remembered up to and including the day before the next scheduled dose. No other clinically important covariates were identified. CONCLUSION: The models in the present analysis adequately described PK and PD characteristics of omarigliptin and supported the dosing and administration section of the omarigliptin label.

Design and synthesis of novel potent anticoagulant and anti-tyrosinase pyranopyrimidines and pyranotriazolopyrimidines: Insights from molecular docking and SAR analysis.

Pyrimidine-fused compounds are of great interest for the discovery of potent bioactive agents. This study describes the synthesis of novel pyranopyrimidines 3a-f and pyranotriazolopyrimidines 4a-d derivatives via the cyclocondensation reaction of α-functionalized iminoether 2, which was obtained from 2-amino-3-cyanopyrane 1, with a series of primary aromatic amines and hydrazides, respectively. Structures of all synthesized compounds were established on the basis of spectroscopic methods including 1H NMR, 13C NMR and ES-HRMS. They were finally tested for their anticoagulant and anti-tyrosinase activities. Significant results have been obtained and the structure-activity relationship (SAR) was discussed with the help of molecular docking analysis.

UPLC-Q-TOF-MS and UPLC-MS/MS methods for metabolism profiles and pharmacokinetics of major compounds in Xuanmai Ganjie Granules.

Xuanmai Ganjie Granules (XMGJ), a widely used Chinese herbal formula in the clinic, is used for treatment of sore throats and coughs. Despite the chemical constituents having been clarifying by our previous studies, both of the metabolism and pharmacokinetic studies of XMGJ are unclear. This study aimed to explore the disposition process of XMGJ in vivo. A sensitive and selective ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) method was developed to analyze the absorbed components and metabolites in rat plasma and urine after oral administration of XMGJ. A total of 42 absorbed components, including 16 prototype compounds and 26 metabolites, were identified or tentatively characterized in rat plasma and urine after oral administration of XMGJ. Moreover, the pharmacokinetic studies of five compounds of XMGJ were investigated using ultra-high liquid chromatography with tandem mass spectrometry method. The results indicated that liquiritin, harpagoside, glycyrrhetic acid, liquiritigenin, formononetin and their metabolites might be the major components involved in the pharmacokinetic and metabolism process of XMGJ. This research showed a comprehensive investigation of XMGJ in vivo, which could provide a meaningful basis for further material basis and pharmacological as well as toxicological research.

Pharmacokinetics of harpagoside in horses after intragastric administration of a Devil's claw (Harpagophytum procumbens) extract.

Devil's claw is used for the treatment of inflammatory symptoms and degenerative disorders in horses since many years, but without the substantive pharmacokinetic data. The pharmacokinetic parameters of harpagoside, the main active constituent of Harpagophytum procumbens DC ex Meisn., were evaluated in equine plasma after administration of Harpagophytum extract FB 8858 in an open, single-dose, two-treatment, two-period, randomized cross-over design. Six horses received a single dose of Harpagophytum extract, corresponding to 5 mg/kg BM harpagoside, and after 7 days washout period, 10 mg/kg BM harpagoside via nasogastric tube. Plasma samples at certain time points (before and 0-24 hr after administration) were collected, cleaned up by solid-phase extraction, and harpagoside concentrations were determined by LC-MS/MS using apigenin-7-glucoside as internal standard. Plasma concentration-time data and relevant parameters were described by noncompartmental model through PKSolver software. Harpagoside could be detected up to 9 hr after administration. Cmax was found at 25.59 and 55.46 ng/ml, t1/2 at 2.53 and 2.32 hr, respectively, and tmax at 1 hr in both trials. AUC0-inf was 70.46 and 117.85 ng hr ml-1 , respectively. A proportional relationship between dose, Cmax and AUC was observed. Distribution (Vz /F) was 259.04 and 283.83 L/kg and clearance (CL/F) 70.96 and 84.86 L hr-1  kg-1 , respectively. Treatment of horses with Harpagophytum extract did not cause any clinically detectable side effects.

A validated LC-MS/MS method for the determination of specnuezhenide and salidroside in rat plasma and its application to a pharmacokinetic study.

The aim of this study was to establish and validate a rapid and sensitive LC-MS/MS method for the simultaneous determination of specnuezhenide and its bioactive metabolite salidroside in rat plasma. Protein precipitation was carried out and the analytes were separated on a Waters Acquity UPLC HSS T3 column (2.1 × 100 mm, 1.8 µm). A mobile phase consisting of acetonitrile and 0.1% formic acid aqueous solution was used for elution under gradient conditions at a flow rate of 0.4 mL/min. Quantification was performed in the negative multiple reaction monitoring mode with precursor-to-product transitions at m/z 685.2 → 453.1 for specnuezhenide, m/z 229.3 → 119.0 for salidroside and 493.2 → 147.1 for the internal standard. The method showed good linearity, accuracy, precision and stability in the range 0.5-500.0 ng/mL for specnuezhenide and salidroside. The values of the matrix effect were within the range of 100.02-111.87% for both analytes, while the mean extraction recovery was within the range 64.19-78.26%. The intra- and inter-day precisions (RSD) were <13.49% and the accuracy (RR) ranged from 93.59 to 102.24%. This study was successfully utilized for the pharmacokinetic study of specnuezhenide in rats after oral and intravenous administration. The oral bioavailability of specnuezhenide was 1.93%.

Ultra-high pressure liquid chromatography-tandem mass spectrometry method for the determination of omarigliptin in rat plasma and its application to a pharmacokinetic study in rats.

Omarigliptin is a novel long-acting dipeptidyl peptidase-4 inhibitor used for the treatment of type 2 diabetes. In this work, a sensitive and selective ultra-high pressure liquid chromatography tandem mass spectrometry method was developed and validated for the determination of omarigliptin in rat plasma. Sample preparation was performed by protein precipitation with acetonitrile. Chromatographic separation of analytes was achieved on an RRHD Eclipse Plus C18 column (2.1 × 50 mm, 1.8 µm), using gradient mobile phase (0.1% formic acid-acetonitrile) at a flow rate of 0.4 mL/min. Detection was performed in multiple reaction monitoring mode, with target fragment ions m/z 399.1 → 152.9 for omarigliptin and m/z 237.1 → 194 for the internal standard. The total run time was 4 min. Retention time of omarigliptin and internal standard was 1.25 and 2.12 min, respectively. Relative standard deviation (%) of the intra- and inter-day precision was below 10.0%, and accuracy was between 97.9% and 105.3%. Calibration curve was established over the range 2-5000 ng/mL with good linearity. The lower limit of quantification and limit of detection of omarigliptin were 2 and 0.25 ng/mL, respectively. Mean recoveries were in the range 87.3-95.1% for omarigliptin. No matrix effect was observed in this method. This novel method has been successfully applied to a pharmacokinetic study of omarigliptin in rats. The absolute bioavailability of omarigliptin was identified as high as 87.31%.

In Vivo Pharmacodynamic Target Investigation of Two Bacterial Topoisomerase Inhibitors, ACT-387042 and ACT-292706, in the Neutropenic Murine Thigh Model against Streptococcus pneumoniae and Staphylococcus aureus.

ACT-387042 and ACT-292706 are two novel bacterial topoisomerase inhibitors with broad-spectrum activity against Gram-positive and -negative bacteria, including methicillin-resistant Staphylococcus aureus and penicillin- and fluoroquinolone-resistant Streptococcus pneumoniae We used the neutropenic murine thigh infection model to characterize the pharmacokinetics (PK)/pharmacodynamics (PD) of these investigational compounds against a group of 10 S. aureus and S. pneumoniae isolates with phenotypic resistance to beta-lactams and fluoroquinolones. The in vitro activities of the two compounds were very similar (MIC range, 0.03 to 0.125 mg/liter). Plasma pharmacokinetics were determined for each compound by using four escalating doses administered by the subcutaneous route. In treatment studies, mice had 10(7.4) to 10(8) CFU/thigh at the start of therapy with ACT-387042 and 10(6.7) to 10(8.3) CFU/thigh at the start of therapy with ACT-292706. A dose-response relationship was observed with all isolates over the dose range. Maximal kill approached 3 to 4 log10 CFU/thigh compared to the burden at the start of therapy for the highest doses examined. There was a strong relationship between the PK/PD index AUC/MIC ratio (area under the concentration-time curve over 24 h in the steady state divided by the MIC) and therapeutic efficacy in the model (R(2), 0.63 to 0.82). The 24-h free-drug AUC/MIC ratios associated with net stasis for ACT-387042 against S. aureus and S. pneumoniae were 43 and 10, respectively. The 24-h free-drug AUC/MIC ratios associated with net stasis for ACT-292706 against S. aureus and S. pneumoniae were 69 and 25, respectively. The stasis PD targets were significantly lower for S. pneumoniae (P < 0.05) for both compounds. The 1-log-kill AUC/MIC ratio targets were ∼2- to 4-fold higher than stasis targets. Methicillin, penicillin, or ciprofloxacin resistance did not alter the magnitude of the AUC/MIC ratio required for efficacy. These results should be helpful in the design of clinical trials for topoisomerase inhibitors.

Targeted and Untargeted Metabolomics to Explore the Bioavailability of the Secoiridoids from a Seed/Fruit Extract (Fraxinus angustifolia Vahl) in Human Healthy Volunteers: A Preliminary Study.

The bark, seeds, fruits and leaves of the genus Fraxinus (Oleaceae) which contain a wide range of phytochemicals, mostly secoiridoid glucosides, have been widely used in folk medicine against a number of ailments, yet little is known about the metabolism and uptake of the major Fraxinus components. The aim of this work was to advance in the knowledge on the bioavailability of the secoiridoids present in a Fraxinus angustifolia Vahl seed/fruit extract using both targeted and untargeted metabolomic analyses. Plasma and urine samples from nine healthy volunteers were taken at specific time intervals following the intake of the extract and analyzed by UPLC-ESI-QTOF. Predicted metabolites such as tyrosol and ligstroside-aglycone glucuronides and sulfates were detected at low intensity. These compounds reached peak plasma levels 2 h after the intake and exhibited high variability among the participants. The ligstroside-aglycone conjugates may be considered as potential biomarkers of the Fraxinus secoiridoids intake. Using the untargeted approach we additionally detected phenolic conjugates identified as ferulic acid and caffeic acid sulfates, as well as hydroxybenzyl and hydroxyphenylacetaldehyde sulfate derivatives which support further metabolism of the secoiridoids by phase I and (or) microbial enzymes. Overall, the results of this study suggest low uptake of intact secoiridoids from a Fraxinus angustifolia Vahl extract in healthy human volunteers and metabolic conversion by esterases, glycosidases, and phase II sulfo- and glucuronosyl transferases to form smaller conjugated derivatives.

[Studies on effects of Achyranthes bidentata on tongsaimai pellets main active ingredients chlorogenic acid, isoliquiritin, harpagoside and glycyrrhizin in vivo pharmacokinetics].

To study on the effects of Achyranthes bidentata on Tongsaimai pellets main active ingredients chlorogenic acid, isoliquiritin, harpagoside and glycyrrhizin in rats in vivo pharmacokinetic behaviors, a method for the simultaneous determination of chlorogenic acid, isoliquiritin, harpagoside and liquiritigenin in rat plasma was established by UPLC-MS/MS. The analysis was performed on a waters Acquity BEH C18 column (2.1 mm x 100 mm, 1.7 microm) with the mixture of acetonitrile and 0.1% formic acid/water as mobile phase, and the gradient elution at a flow rate of 0.3 mL x min(-1). The analytes were detected by tandem mass spectrometry with the electrospray ionization (ESI) source and in the multiple reaction monitoring (MRM) mode. It turned out that the analytes of Tongsaimai pellets groups C(max) and AUC(Q-infinity) values were higher than that with A. bidentata group, and the C(max) values of chlorogenic acid had significantly difference (P < 0.05), the AUC(0-infinity) values of chlorogenic acid and glycyrrhizin had significantly difference (P < 0.05); The T(max) and CL values of two groups had no significantly difference. Results showed that the established method was specific, rapid, accurate and sensitive for the studies of Tongsaimai pellets four main active ingredients in rat in vivo pharmacokinetic, and A. bidentata have varying degrees of effects on Tongsaimai pellets four main active ingredients in rat in vivo pharmacokinetic behaviors.

Validated LC-MS method for simultaneous quantitation of catalpol and harpagide in rat plasma: application to a comparative pharmacokinetic study in normal and diabetic rats after oral administration of Zeng-Ye-Decoction.

A simple and efficient liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for simultaneous quantitation of catalpol and harpagide in normal and diabetic rat plasma. Protein precipitation extraction with acetonitrile was carried out using salidroside as the internal standard (IS). The LC separation was performed on an Elite C18 column (150 × 4.6 mm, 5 µm) with the mobile phase consisting of acetonitrile and water within a runtime of 12.0 min. The analytes were detected without endogenous interference in the selected ion monitoring mode with positive electrospray ionization. Calibration curves offered satisfactory linearity (r > 0.99) at linear range of 0.05-50.0 µg/mL for catalpol and 0.025-5.0 µg/mL for harpagide with the lower limits of quantitation of 0.05 and 0.025 µg/mL, respectively. Intra- and inter-day precisions (RSD) were <9.4%, and accuracy (RE) was in the -6.6 to 4.9% range. The extraction efficiencies of catalpol, harpagide and IS were all >76.5% and the matrix effects of the analytes ranged from 86.5 to 106.0%. The method was successfully applied to the pharmacokinetic study of catalpol and harpagide after oral administration of Zeng-Ye-Decoction to normal and diabetic rats, respectively.

[Absorption and pharmacokinetics of radix rehmanniae in rats].

In this paper, absorption and pharmacokinetic study of Radix Rehmanniae was studied by liquid chromatography coupled with mass spectrometry method after oral administration to rats. By comparing the chromatograms of ultraviolet, full scan, extracted ion and selective reaction monitoring (SRM) of standard solution, Radix Rehmanniae, blank plasma and rat plasma post drug administration, catalpol and ajugol were found to be the main compounds absorbed from Radix Rehmanniae. Plasma concentrations of aucubin, dihydrocatalpol, rehmannioside A (or rehmannioside B/ melittoside) and rehmannioside D were very low. Quantitative method for catalpol and aucubin and semi-quantitative method for other compounds in rat plasma were established. The pharmacokinetic study of those absorbed components was conducted after oral administration of 6 g x kg(-1) Radix Rehmanniae water extract to rats. Cmax, t(1/2) and AUC(0-infinity) of catalpol and ajugol were (2349.05 +/- 1438.34) and (104.25 +/- 82.05) ng x mL(-1), (0.86 +/- 0.32) and (0.96 +/- 0.37) h, (4407.58 +/- 2734.89) and (226.66 +/- 188.38) ng x h x mL(-1), respectively. tmax was at 1.00 h for catalpol and ajugol. Both catalpol and ajugol were absorbed and excreted rapidly.

Comparison of the oral bioavailability and tissue disposition of monensin and salinomycin in chickens and turkeys.

The current study describes the pharmacokinetic parameters of two carboxylic polyether ionophores: monensin in turkeys and salinomycin in chickens. These data can be used to understand and predict the occurrence of undesirable residues of coccidiostats in edible tissues of these animal species. Special attention is paid to the distribution of residues between the different edible tissues during and at the end of the treatment period. For the bioavailability studies, monensin was administered to turkeys intravenously, in the left wing vein, at a dose of 0.4 mg /kg and orally at a dose of 20 mg /kg. Salinomycin was administered to chickens intravenously, in the left wing vein, at a dose of 0.25 mg /kg and orally at a dose of 2.5 mg /kg. Residue studies were carried out with supplemented feed at the rate of 100 mg /kg of feed for monensin in turkeys and 70 mg /kg for salinomycin in chickens, respectively. Coccidiostats had a low bioavailability in poultry (around 30% for monensin in chickens, around 1% for monensin in turkeys and around 15% for salinomycin in chickens). Monensin in chickens had a longer terminal half-life (between 3.07 and 5.55 h) than both monensin in turkeys (between 1.36 and 1.55 h) and salinomycin in chickens (between 1.33 and 1.79 h). The tissue /plasma partition coefficients showed a higher affinity of both monensin and salinomycin for fat, followed by liver and muscle tissue. The depletion data showed a fairly rapid elimination of coccidiostats in all the tissues after cessation of treatment. According to the results of depletion studies, a withdrawal period of 1 day seems sufficient to avoid undesirable exposure of consumers.

Identification and quantification of salinomycin in intoxicated human plasma by liquid chromatography-electrospray tandem mass spectrometry.

Salinomycin is a polyether ionophore antibiotic that is widely used in poultry and livestock. Exposure of humans to salinomycin via inhalation or ingestion can cause severe toxicity. The aim of the present work was to develop a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the rapid identification and quantification of salinomycin in human plasma. After removing protein using methanol, plasma samples were eluted from a Waters Xterra(®) MS C18 column with an isocratic mobile phase. Detection and quantification of the drug were performed with a triple-quadruple mass spectrometer by monitoring for two specific transitions in the electrospray, positive-ion, multiple-reaction monitoring mode. Assay validation showed good linearity (r(2) = 0.998). The detection and quantification limits of the method were 0.6 and 16 pg/mL, respectively. The inter- and intraday coefficients of variation for the assay were both <15%. Twelve authentic plasma samples from intoxicated patients were analyzed using this method. Salinomycin was detected in six samples, at concentrations of between 0.6 and 46.5 pg/mL. The described assay method allows the sensitive and rapid identification and quantification of salinomycin in human plasma, and thus provides a valuable tool for the specific diagnosis of salinomycin intoxication in clinical and emergency rescue practice.

Simultaneous quantification of oleuropein and its metabolites in rat plasma by liquid chromatography electrospray ionization tandem mass spectrometry.

Oleuropein (OE) is the cardinal bioactive compound derived from Olea europaea and possesses numerous beneficial properties for human health. However, despite the plethora of analytical methods that have studied the biological fate of olive oil-derived bioactive compounds, no validated methodology has been published to date for the simultaneous determination of OE, along with all its major metabolites. In this study, a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI MS/MS) method has been developed and validated for the quantification of OE, simultaneously with its main metabolites hydroxytyrosol, 2-(3,4-dihydroxyphenyl)acetic acid, 4-(2-hydroxyethyl)-2-methoxy-phenol or homovanillyl alcohol, 2-(4-hydroxy-3-methoxyphenyl)acetic acid or homovanillic acid, and elenolic acid in rat plasma matrix. Samples were analyzed by LC-ESI MS/MS prior to and after enzymatic treatment. A solid-phase extraction step with high mean recovery for all compounds was performed as sample pretreatment. Calibration curves were linear for all bioactive compounds over the range studied, while the method exhibited good accuracy, intra- and inter-day precision. The limit of detection was in the picogram range (per milliliterof plasma) for HT and OE and in the nanogram range (per milliliter of plasma) for the other analytes, and the method was simple and rapid. The developed methodology was successfully applied for the simultaneous quantification of OE and its aforementioned metabolites in rat plasma samples, thus demonstrating its suitability for pharmacokinetics, as well as bioavailability and metabolism studies.

Modulation of the partition coefficient between octanol and buffer at pH 7.4 and pKa to achieve the optimum balance of blood clearance and volume of distribution for a series of tetrahydropyran histamine type 3 receptor antagonists.

The relationship between rat pharmacokinetics and physicochemical parameters [the partition coefficient between octanol and buffer at pH 7.4 (log D((7.4))) and pK(a)] was studied for a series of tetrahydropyran compounds. Sixteen compounds ranging in log D((7.4)) 0.1 to 1.8 were administered intravenously to rats, and the pharmacokinetic parameters were determined from blood concentration time curves. Across the series, a weak correlation was observed between log D((7.4)) and blood clearance, suggesting that log D((7.4)) values less than 0.5 were required to prevent clearance at hepatic blood flow. In terms of the volume of distribution (V(d)), the compounds fell into three distinct subseries characterized by the number of basic centers and differences in ionization of each basic center at physiological pH. These were referred to as the monobasic, weak second base, and strong second base subseries. All the compounds exhibited V(d) greater than body water, as would be expected from their lipophilic and basic nature. For a given clog P, the strong second base subseries showed higher V(d) than the weak second base subseries, which in turn exhibited higher values than the monobasic subseries. In addition, for the weak second base subseries, V(d) could be tuned by modulating the pK(a) of the second basic center. This relationship was rationalized in respect to the interactions of the ionizable centers with phospholipid heads in the cell membrane and/or lysosomal trapping. Compounds in the weak second base subseries showed optimal V(d), and when combined with a log D((7.4)) of 0.1, driving to moderate blood clearance, one compound showed the optimal pharmacokinetic profile.

Optimization of solid-phase extraction for the liquid chromatography-mass spectrometry analysis of harpagoside, 8-para-coumaroyl harpagide, and harpagide in equine plasma and urine.

Solid-phase extraction cartridges among those usually used for screening in horse doping analyses are tested to optimize the extraction of harpagoside (HS), harpagide (HG), and 8-para-coumaroyl harpagide (8PCHG) from plasma and urine. Extracts are analyzed by liquid chromatography coupled with multi-step tandem mass spectrometry. The extraction process retained for plasma applies BondElut PPL cartridges and provides extraction recoveries between 91% and 93%, with RSD values between 8 and 13% at 0.5 ng/mL. Two different procedures are needed to extract analytes from urine. HS and 8PCHG are extracted using AbsElut Nexus cartridges, with recoveries of 85% and 77%, respectively (RSD between 7% and 19%). The extraction of HG involves the use of two cartridges: BondElut PPL and BondElut C18 HF, with recovery of 75% and RSD between 14% and 19%. The applicability of the extraction methods is determined on authentic equine plasma and urine samples after harpagophytum or harpagoside administration.