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Cystic fibrosis is one of the most common genetic diseases among caucasian population. This disease is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding for the CFTR protein. Lumacaftor, elexacaftor, tezacaftor, and ivacaftor were currently used as the treatment to Cystic fibrosis. In this study, we describe a new method for the simultaneous quantification of four molecules: lumacaftor, elexacaftor, tezacaftor, and ivacaftor, alongside two metabolites of ivacaftor, specifically hexyl-methyl ivacaftor and ivacaftor carboxylate by liquid chromatography-tandem mass spectrometry. This method holds significant utility for therapeutic drug monitoring and the optimization of treatments related to CFTR modulators. Molecules were extracted from 100 µL of plasma by a simple method of protein precipitation using acetonitrile. Following extraction, chromatographic separation was carried out by reverse chromatography on a C18 analytical column, using a gradient elution of water (0.05 % formic acid, V/V) and acetonitrile (0.05 % formic acid, V/V). The run time was 7 minutes at a flow rate of 0.5 mL/min. After separation, molecules were detected by electrospray ionization on a Xevo TQD triple-quadrupole-mass-spectrometer (Waters®, Milford, USA). The calibration range were: 0.053-20.000 mg/L for elexacaftor, tezacaftor and lumacaftor, 0.075-14.000 mg/L for ivacaftor, and 0.024-6.500 mg/L for hexyl-methyl ivacaftor and ivacaftor carboxylate. The proposed method underwent throughout validation demonstrating satisfactory precision (inter- and intra-day coefficients of variation less than 14.3 %) and a good accuracy (inter- and intra-day bias ranging between -13.7 % and 14.7 %) for all the analytes. The presented method for the simultaneous quantification of CFTR modulators and their metabolites in human plasma has undergone rigorous validation process yielding good results including strong precision and accuracy for all analytes. This method has been effectively used in routine analytical analysis and clinical investigations within our laboratory., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Therapeutic drug monitoring (TDM) of elexacaftor, tezacaftor, ivacaftor (ETI) could be a useful tool to increase efficacy and decrease the risk of adverse effects in people with Cystic Fibrosis (pwCF). It is however unclear whether drug exposure should be monitored by assessment of trough (C min ) levels or determination of the area under the curve (AUC). Hence, in this study the correlation between measured C min concentration and AUC was evaluated. Serial plasma samples, including C min , were drawn after administration of ETI in order to calculate the AUC and assess the correlation between the two parameters. A linear correlation between C min and AUC 0-24h was found, with Pearson's r correlation coefficients of 0.963, 0.908 and 0.860 for elexacaftor, tezacaftor and ivacaftor, respectively. Exposure of ETI may be monitored by assessment of C min levels., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2024. Published by Elsevier B.V.)

Oxidative stress and inflammation have been considered the main factors in the liver injury of clofibrate (CF). To obtain a novel antihyperlipidemic agent with antioxidant, anti-inflammation and hepatoprotection, the combination of sesamol and clofibric acid moieties was performed and achieved sesamol-clofibrate (CF-Sesamol). CF-Sesamol showed significant hypolipidemia effects in hyperlipidemia mice induced by Triton WR 1339, reducing TG by 38.8% (P < 0.01) and TC by 35.1% (P < 0.01). CF-Sesamol also displayed an alleviating effect on hepatotoxicity. The hepatic weight and hepatic coefficient were decreased. The amelioration of liver function was observed, such as aspartate and lactate transaminases (AST and ALT), alkaline phosphatase (ALP) and total proteins (TP) levels. Liver histopathological examination showed that hepatocyte necrosis, cytoplasmic loosening, nuclear degeneration and inflammatory cell infiltration reduced obviously by treatment with CF-Sesamol. Related molecular mechanisms on hepatoprotection showed that CF-Sesamol up-regulated Nrf2 and HO-1 expression and down-regulated p-NF-κB p65 expression in hepatic tissues. CF-Sesamol has significant antioxidant and anti-inflammatory effects. Plasma antioxidant enzymes such as SOD and CAT increased, anti-lipid peroxidation product MDA decreased. The expression of TNF-α and IL-6 inflammatory cytokines in liver was significantly lower than that in the CF group. The results indicated that CF-Sesamol exerted more potent antihyperlipidemic effects and definite hepatoprotective activity partly through the Nrf2/NF-κB-mediated signaling pathway., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Scope: Processed meat intake has been associated with adverse health outcomes. However, little is known about the type of processed meat more particularly responsible for these effects. This study aims to identify novel biomarkers for processed meat intake., Methods and Results: In a controlled randomized cross-over dietary intervention study, 12 healthy volunteers consume different processed and non-processed meats for 3 consecutive days each. Metabolomics analyses are applied on post-intervention fasting blood and urine samples to identify discriminating molecular features of processed meat intake. Nine and five pepper alkaloid metabolites, including piperine, are identified as major discriminants of salami intake in urine and plasma, respectively. The associations with processed meat intake are tested for replication in a cross-sectional study (n = 418) embedded within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. Three of the serum metabolites including piperine are associated with habitual intake of sausages and to a lesser extent of total processed meat., Conclusion: Pepper alkaloids are major discriminants of intake for sausages that contain high levels of pepper used as ingredient. Further work is needed to assess if pepper alkaloids in combination with other metabolites may serve as biomarkers of processed meat intake., (© 2021 Wiley-VCH GmbH.)

Herbals that are widely consumed as therapeutic alternatives to conventional drugs for cardiovascular diseases, may lead to herb-drug interactions (HDIs). Atorvastatin (ATR) is drug of choice for hyperlipidemia and is extensively metabolized through CYP3A4 enzyme. Thus, we postulate that concomitant administration of ATR with piperine (PIP, potent inhibitor of CYP3A4 enzyme)/ridayarishta (RID, cardiotonic herbal formulations containing PIP) may lead to potential HDI. A simple, accurate, sensitive high-performance liquid chromatography-photodiode array detection method using Kromasil-100 C18 column, mobile phase acetonitrile: 30 mM phosphate buffer (55:45 v/v) pH 4.5 with flow rate gradient programming was developed to study the potential HDI in rats. Method was found to be linear (2-100 ng/mL) with Lower Limit of Detection (LLOD) 2 ng/mL. The precision (%CV < 15%), accuracy (-1.0 to -10% R.E) with recoveries above 90% from rat plasma of ATR and IS were obtained. The pharmacokinetic (PK) interactions studies on co-administration of ATR (8.4 mg/kg, p.o.) with PIP (35 mg/kg, p.o.), demonstrated a threefold increase in Cmax of ATR (P < 0.01) with significant increase in AUC0-t/AUC0-∞ compared to ATR alone indicating potential PK-HDI. However co-administration of RID (4.2 mL/kg, p.o.) showed less significant changes (P > 0.05) indicating low HDI. The pharmacodynamic effects/interactions study (TritonX-100 induced hyperlipidemic model in rats) suggested no significant alterations in the lipid profile on co-administration of PIP/RID with ATR, indicating that there may be no significant pharmacodynamic interactions., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Widely accessible food phytochemicals such as curcumin have been reported to have anti-inflammatory and anticarcinogenic properties. However, curcumin has poor absorption in the gut, and piperine has been of interest as a dietary compound that can enhance curcumin bioavailability. The aim of this study was to develop and optimize a technique using reversed-phase chromatography with multi-wavelength detection for the simultaneous measurement of curcumin and piperine in various biological matrices. Emodin was used as an internal standard. Protein precipitation and liquid-liquid extraction based on acetonitrile provided good recovery of these analytes. A 150 mm × 4.6 mm I.D. Luna C18 column was used under isocratic conditions to separate curcumin, piperine, and emodin with baseline resolution, and with good separation from other sample components, in as little as 4 min. The detection limits for curcumin and piperine were 3 and 7 ng/mL, respectively. This method has been used to quantitate these compounds in samples such as human intestinal epithelial cell lysates and mouse plasma or GI tissues in research aimed at examining the bioavailability of curcumin in the presence of piperine., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Diabetic foot ulcer is an intractable complication of diabetes, characterized by the disturbed inflammatory and proliferative phases of wound healing. Sesamol, a phenolic compound, has been known for its powerful antioxidant, anti-inflammatory, anti-hyperglycaemic and wound healing properties. The aim of the present study was to develop a sesamol nano formulation and to study its effect on the various phases of the wound healing process in diabetic foot condition., Methods: Sesamol-PLGA (SM-PLGA) nanosuspension was developed  using nanoprecipitation method. TEM, in vitro drug release assay and in vivo pharmacokinetic studies were performed for the optimised formulation. Diabetic foot ulcer (DFU) in high fat diet (HFD)-fed streptozotocin-induced type-II diabetic animal model was used to assess the SM-PLGA nanosuspension efficacy. SM-PLGA nanosuspension was administered by oral route. TNF-α levels were estimated using ELISA and Western blot analysis was performed to assess the effect on the expression of HSP-27, ERK, PDGF-B and VEGF in wound tissue. Wound re-epithelization, fibroblast migration, collagen deposition and inflammatory cell infiltration were assessed by H&E and Masson's trichrome staining. Effect on angiogenesis was assessed by CD-31 IHC staining in wound sections., Results: The optimized SM-PLGA nanosuspension had an average particle size of <300 nm, PDI<0.200 with spherical shaped particles. Approximately 80% of the drug was released over a period of 60 h in in vitro assay. Half-life of the formulation was found to be 13.947 ± 0.596 h. SM-PLGA nanosuspension treatment decreased TNF-α levels in wound tissue and accelerated the collagen deposition. Whereas, HSP-27, ERK, PDGF-B and VEGF expression increased and improved new blood vessels' development. Rapid re-epithelization, fibroblast migration, collagen deposition and reduced inflammatory cell infiltration at the wound site were also observed., Conclusion: Results indicate that sesamol-PLGA nanosuspension significantly promotes the acceleration of wound healing in diabetic foot ulcers by restoring the altered wound healing process in diabetic condition., Competing Interests: The authors report no conflicts of interest for this work., (© 2020 Gourishetti et al.)

W34 is a prodrug of FL118, and it can be converted to FL118 via a hydrolysis reaction. In this report, a highly sensitive LC-MS/MS method using a C 18 column was validated and used for the simultaneous determination of W34 and FL118 in rat blood. A stepwise gradient elution with 0.1% formic acid in water and acetonitrile was employed. The assays were linear over a concentration range of 0.50-50.0 ng/ml for both W34 and FL118. The accuracy of the validation method ranged from 89.74 to 98.94% for W34 and from 88.61 to 94.60% for FL118. The precision was within 7.15% for W34 and 9.63% for FL118. Extraction recoveries of W34 were 94.56-100.49 and 87.67-106.32% for FL118. No significant matrix effects for both W34 and FL118 were observed in blood. The assay has been successfully applied to biological samples obtained from a stability and pharmacokinetic study of W34 and FL118., (© 2020 John Wiley & Sons, Ltd.)

Piperine (Pip) has been widely studied for its multiple activities such as antidepressant, anti-epileptic, and so forth. However, the poor water solubility coupled with low bioavailability may inevitably hinder the application of Pip in the clinical setting. In this study, a formulation strategy was proposed to spontaneously resolve the low bioavailability and dose dividing issue of Pip. The matrix pellets (Pip-SR-pellets) consisting of Pip solid dispersion (Pip-SD) and hydroxypropylmethyl cellulose-K100 were developed to achieve an increased and sustained release profile in vitro . The Pip-SR-pellets were compacted into fast disintegrating tablets (FDTs) with a blend of excipients comprising lactose, MCC, LS-HPC, and CMS-Na. The Pip-SD was characterized by solubility study and XRD. The evaluation of the cross-sectional morphology of the Pip-FDTs via scanning electron microscope proved that Pip-SR-pellets maintained its structural integrity during compression and were uniformly distributed in the Pip-FDTs. The release profile of Pip-SR-pellets was highly consistent with the Pip-FDTs. In vivo pharmacokinetics study demonstrated that the relative bioavailability of Pip-SR-pellets was approximately 2.70-fold higher than that of the pure drug, and 1.62-fold compared with that of Pip-SD. This work therefore showed a potential industrialized method could be applied to formulate poorly water-soluble drug that has dose-dividing requirement.

Obesity is one of the major risk factor for cardiovascular and metabolic diseases. A disproportional accumulation of fat at visceral (VAT) compared to subcutaneous sites (SAT) has been suspected as a key detrimental event. We used non-targeted metabolomics profiling to reveal metabolic pathways associated with higher VAT or SAT amount among subjects free of metabolic diseases to identify possible contributing metabolic pathways. The study population comprised 491 subjects [mean (standard deviation): age 44.6 yrs (13.0), body mass index 25.4 kg/m² (3.6), 60.1% females] without diabetes, hypertension, dyslipidemia, the metabolic syndrome or impaired renal function. We associated MRI-derived fat amounts with mass spectrometry-derived metabolites in plasma and urine using linear regression models adjusting for major confounders. We tested for sex-specific effects using interactions terms and performed sensitivity analyses for the influence of insulin resistance on the results. VAT and SAT were significantly associated with 155 (101 urine) and 49 (29 urine) metabolites, respectively, of which 45 (27 urine) were common to both. Major metabolic pathways were branched-chain amino acid metabolism (partially independent of insulin resistance), surrogate markers of oxidative stress and gut microbial diversity, and cortisol metabolism. We observed a novel positive association between VAT and plasma levels of the potential pharmacological agent piperine. Sex-specific effects were only a few, e.g. the female-specific association between VAT and O-methylascorbate. In brief, higher VAT was associated with an unfavorable metabolite profile in a sample of healthy, mostly non-obese individuals from the general population and only few sex-specific associations became apparent.

Background: Alzheimer's disease (AD) is a complex neurological disorder with contributions from genetic and environmental factors. High-resolution metabolomics (HRM) has the potential to identify novel endogenous and environmental factors involved in AD. Previous metabolomics studies have identified circulating metabolites linked to AD, but lack of replication and inconsistent diagnostic algorithms have hindered the generalizability of these findings. Here we applied HRM to identify plasma metabolic and environmental factors associated with AD in two study samples, with cerebrospinal fluid (CSF) biomarkers of AD incorporated to achieve high diagnostic accuracy., Methods: Liquid chromatography-mass spectrometry (LC-MS)-based HRM was used to identify plasma and CSF metabolites associated with AD diagnosis and CSF AD biomarkers in two studies of prevalent AD (Study 1: 43 AD cases, 45 mild cognitive impairment [MCI] cases, 41 controls; Study 2: 50 AD cases, 18 controls). AD-associated metabolites were identified using a metabolome-wide association study (MWAS) framework., Results: An MWAS meta-analysis identified three non-medication AD-associated metabolites in plasma, including elevated levels of glutamine and an unknown halogenated compound and lower levels of piperine, a dietary alkaloid. The non-medication metabolites were correlated with CSF AD biomarkers, and glutamine and the unknown halogenated compound were also detected in CSF. Furthermore, in Study 1, the unknown compound and piperine were altered in MCI patients in the same direction as AD dementia., Conclusions: In plasma, AD was reproducibly associated with elevated levels of glutamine and a halogen-containing compound and reduced levels of piperine. These findings provide further evidence that exposures and behavior may modify AD risks., (© 2019 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association.)

Background: Oxyresveratrol is a major bioactive component derived from the heartwood of Artocarpus lacucha. This compound exerts several biological activities, including neuroprotective effects in vitro and in vivo. However, there is limited pharmacokinetic information on this compound, especially its distribution in neuronal tissue and its route of excretion. The aim of this study was to investigate the pharmacokinetic profiles of oxyresveratrol alone and in combination with piperine as a bioenhancer in rats., Methods: Male Wistar rats were administered with oxyresveratrol 10 mg/kg, oxyresveratrol 10 mg/kg plus piperine 1 mg/kg via intravenous or oxyresveratrol 100 mg/kg, oxyresveratrol 100 mg/kg plus piperine 10 mg/kg via oral gavage. Plasma, internal organs, urine, and feces were collected. Determination of the oxyresveratrol concentration in biological samples was performed by liquid chromatography tandem mass spectrometry., Results: The combination with piperine had shown a significantly higher maximum concentration in plasma approximately 1500 μg/L within 1-2 h after oral dosing, and could increase oral bioavailability of oxyresveratrol approximately 2-fold. Oxyresveratrol could widely distributed most of the internal organs with a tissue to plasma ratio of 10-100 fold within 5 min after dosing. Urinary excretion of oxyresveratrol glucuronide was the major route of excretion after administration of oxyresveratrol alone and in combination with piperine., Conclusion: The addition of piperine could enhance some of the pharmacokinetic properties of oxyresveratrol via both intravenous and oral administration. This pharmacokinetic information will be useful for appropriate strategies to develop oxyresveratrol as a phytopharmaceutical product.

Resveratrol and piperine are proven for their therapeutic benefits to treat various diseases. Due to their synergistic actions and combined drug delivery application, a rapid and specific RP-HPLC method was developed and validated as per ICH guidelines, by using an isosbestic point. The chromatographic separation was performed with Luna 5 μ 100 Å C-18(2) HPLC column by using acetonitrile (ACN): phosphate buffer (0.01% orthophosphoric acid) (55:45) as mobile phase, at 1 mL/min of flow rate and 330 nm. The developed method was found to be linear over the concentration range of 0.25-8 μg/mL with correlation coefficient value >0.999. The developed method was accurate (percent recovery 98.06-101.74%), precise (percent relative standard deviation <2.0%), and robust. The limit of detection and limit of quantification for resveratrol were found to be 0.02 and 0.08 μg/mL, respectively and 0.04 and 0.11 μg/mL, for piperine, respectively. The developed method was also validated in human plasma as per ICH guidelines. Moreover, stress degradation studies of both phytoconstituents were studied and the relevancy of the developed method was analyzed on cubosome nanoformulation. A good separation of drug peaks was observed in the presence of the degradation products. This method could thus be used for regular in vitro and in vivo estimation of piperine and resveratrol., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Drug-drug interaction (DDI) studies are described for tezacaftor/ivacaftor, a new cystic fibrosis transmembrane conductance regulator modulator therapy for the treatment of cystic fibrosis. Three phase I DDI studies were conducted in healthy subjects to characterize the DDI profile of tezacaftor/ivacaftor with cytochrome P450 (CYP)3A substrates, CYP3A inhibitors, and a permeability glycoprotein (P-gp) substrate. The effects of steady-state tezacaftor/ivacaftor on the pharmacokinetics (PKs) of digoxin (a P-gp substrate), midazolam, and ethinyl estradiol/norethindrone (CYP3A substrates) were evaluated. Effects of strong (itraconazole) and moderate (ciprofloxacin) CYP3A inhibitors on tezacaftor/ivacaftor PKs were also determined. Tezacaftor/ivacaftor increased digoxin area under the curve (AUC) by 30% but did not affect midazolam, ethinyl estradiol, or norethindrone exposures. Itraconazole increased the AUC of tezacaftor 4-fold and ivacaftor 15.6-fold. Ciprofloxacin had no significant effect on tezacaftor or ivacaftor exposure. Coadministration of tezacaftor/ivacaftor may increase exposure of sensitive P-gp substrates. Tezacaftor/ivacaftor is unlikely to impact exposure of drugs metabolized by CYP3A, including hormonal contraceptives. Strong CYP3A inhibitors significantly increase the exposures of tezacaftor and ivacaftor., (© 2019 Vertex Pharmaceuticals, Incorporated. Clinical and Translational Science published by Wiley Periodicals, Inc. on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

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 [ 3 H] 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 (IC 50  = 37 nM), NET (IC 50  = 105 nM) and SERT (IC 50  = 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., (© 2018 John Wiley & Sons, Ltd.)

With the growing class of CFTR modulator therapy available to more patients and with increasing pregnancies in individuals with CF, there is a growing need to understand the effects of these agents during pregnancy. There are few reports of their continued use in the literature, although it is likely that this is not an uncommon occurrence. We report the uncomplicated and successful pregnancy of a woman treated with lumacaftor/ivacaftor, as well as the clinical course of the infant during the first 9 months of life. We also report drug levels in plasma from the mother, cord blood, breast milk, and infant to estimate fetal and infant drug exposure., (Copyright © 2018 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

N-ethyl pentylone (ephylone) has been identified as the most recent novel stimulant to emerge into the arena of evolving novel psychoactive substances (NPS). Due to its novelty, information regarding case reports with associated quantitative confirmations, biotransformation pathways, and identified unique metabolites will assist the scientific community in understanding the implications of the emergence and risks associated with N-ethyl pentylone use. Authentic blood specimens (n = 26) submitted as part of toxicological death investigations or drugged driving casework tested positive for N-ethyl pentylone, and were quantitatively analyzed by liquid chromatography tandem mass spectrometry (LC-MS-MS). N-ethyl pentylone concentrations ranged from 12 to 1,200 ng/mL, with mean (±standard deviation) and median concentrations of 313 (±366) and 125 ng/mL, respectively, excluding one case measured at 50,000 ng/mL. N-ethyl pentylone was often found in combination with other drugs of abuse and NPS, include a variety of novel opioids including fentanyl analogs. Oral fluid specimens (n = 5), collected from recreational drug users at a dance music festival, were quantitatively analyzed using LC-MS-MS. Concentrations ranged from 12.6 to 1,377 ng/mL. Additional analysis was performed to characterize the metabolic profile of N-ethyl pentylone using human liver microsomes (HLM), followed by confirmation of the presence of the proposed metabolites in a subset of the blood specimens and oral fluid specimens. Metabolomic analysis was performed using a liquid chromatograph quadrupole time-of-flight mass spectrometer (LC-QTOF), followed by data processing using MetabolitePilot™ software. In vivo verification of in vitro HLM-generated metabolites resulted in the confirmation of four metabolites. Reduction of the beta-ketone to an alcohol resulted in the most prominent metabolite found in the authentic specimens, and its uniqueness to N-ethyl pentylone leads to this metabolite being an appropriate biomarker to determine N-ethyl pentylone ingestion. This is the first study to report N-ethyl pentylone concentrations and to characterize the metabolic profile of N-ethyl pentylone.

Hsp90 inhibitors, well studied in the laboratory and clinic for antitumor indications, have promising activity against protozoan pathogens, including Trypanosoma brucei which causes African sleeping sickness, and the malaria parasite, Plasmodium falciparum To progress these experimental drugs toward clinical use, we adapted an in vitro dynamic hollow-fiber system and deployed artificial pharmacokinetics to discover the driver of their activity: either concentration or time. The activities of compounds from three major classes of Hsp90 inhibitors in development were evaluated against trypanosomes. In all circumstances, the activities of the tested Hsp90 inhibitors were concentration driven. By optimally deploying the drug to match its kinetic driver, the efficacy of a given dose was improved up to 5-fold, and maximal efficacy was achieved with a significantly lower drug exposure. The superiority of concentration-driven regimens was evident in vitro over several logs of drug exposure and was predictive of efficacy in a mouse model of African trypanosomiasis. In studies with P. falciparum , antimalarial activity was similarly concentration driven. This experimental strategy offers an expedient and versatile translational tool to assess the impact of pharmacokinetics on antiprotozoal activity. Knowing kinetic governance early in drug development provides an additional metric for judging lead compounds and allows the incisive design of animal efficacy studies., (Copyright © 2018 American Society for Microbiology.)

Background Molecular chaperone targeting has shown promise as a therapeutic approach in human cancers of various histologies and genetic backgrounds. The purine-scaffold inhibitor PU-H71 (NSC 750424), selective for Hsp90 in epichaperome networks, has demonstrated antitumor activity in multiple preclinical cancer models. The present study was a first in-human trial of PU-H71 aimed at establishing its safety and tolerability and characterizing its pharmacokinetic (PK) profile on a weekly administration schedule in human subjects with solid tumors refractory to standard treatments. Methods PU-H71 was administered intravenously over 1 h on days 1 and 8 of 21-day cycles in patients with refractory solid tumors. Dose escalation followed a modified accelerated design. Blood and urine were collected during cycles 1 and 2 for pharmacokinetics analysis. Results Seventeen patients were enrolled in this trial. Grade 2 and 3 adverse events were observed but no dose limiting toxicities occurred, thus the human maximum tolerated dose was not determined. The mean terminal half-life (T 1/2 ) was 8.4 ± 3.6 h, with no dependency to dose level. A pathway for the metabolic disposal of PU-H71 in humans was derived from microsome studies. Fourteen patients were also evaluable for clinical response; 6 (35%) achieved a best response of stable disease for >2 cycles, with 2 patients remaining on study for 6 cycles. The study closed prematurely due to discontinuation of drug supply. Conclusions PU-H71 was well tolerated at the doses administered during this study (10 to 470 mg/m 2 /day), with no dose limiting toxicities.

The new psychoactive substance 3,4-methylenedioxypyrovalerone (MDPV) belongs to the group of synthetic cathinones and is purchased mainly as "research chemical" or "bath salt" on the illegal drug market, also in South Bavaria. MDPV was detected in blood and urine samples from 2010 on in 50 authentic routine cases in a forensic setting. Plasma concentrations in 46 cases with available blood specimens ranged from approximately 1.0 to 301μg/L (median 23.7; mean 47.9μg/L), detected by a fully validated LC-MS/MS method. Subjects aged between 16 and 54 years (median 36; mean 35 years) and reflected experienced chronic drug users. Accused offences were mainly violent crimes such as bodily harm, robberies, homicides and acts of resistance. A lot of subjects showed highly aggressive and violent behavior with endangerment of self and others and/or psychotic symptoms as confusion, hallucinations or paranoia. The risk for such behavior rises with MDPV plasma concentrations above as low as 30μg/L, whereby a time interval of 1.5h on average between incident and/or observation of impairment and blood sampling has to be taken into account. Comprehensive toxicological analysis proved poly-drug use in almost all cases including opiates/opioids, benzodiazepines and other sedatives, antidepressants and other stimulants, also other new psychoactive substances. Alcohol was detected only in three cases. Co-consumed benzodiazepines seem not be able to completely prevent psychotic effects despite their use as first-line treatment for patients with synthetic cathinone poisonings. The study demonstrates that relatively low plasma concentrations of MDPV could be associated with mental impairment which is relevant in the assessment of forensic cases., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 µm, C8 100 Å; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

The stimulant designer drug 3,4-methylenedioxypyrovalerone (MDPV) was first synthesized by Boehringer Ingelheim in 1969 and introduced on the black market in 2006. Only a small number of fatal intoxication cases have been reported in the literature, all with significant blood MDPV concentrations. In this report, we describe one fatality attributed to an idiosyncratic reaction to MDPV. The victim displayed agitation, violent behavior and delirium followed by cardiac arrest. Hyperthermia was observed at the hospital. The MDPV cardiac and femoral blood concentrations were 6 ng/mL. The presence of excited delirium syndrome and MDPV, a drug with a pharmacology similar to cocaine, leads to the conclusion that the victim suffered a fatal adverse reaction to MDPV. This is the first published case of idiosyncratic reaction to MDPV.

Peperomin E (PepE), a naturally occurring secolignan isolated from Peperomia dindygulensis, has drawn much attention recently owing to its anticancer and DNA methyltransferase 1 (DNMT1) inhibitory activity. Here, a simple and sensitive ultra-fast liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of PepE in rat plasma for the first time. Samples were prepared by simple protein precipitation. Separation was performed on an XBridge™ C 18 column using a mobile phase of acetonitrile and 0.1% (v/v) aqueous formic acid. PepE and the internal standard arctigenin were detected in a positive-ion mode using multiple reaction monitoring of the transitions at m/z 413.2 → 261.0 and 373.2 → 137.2, respectively. The calibration curve for PepE was linear over the range of concentrations of 1.46-6000 ng/mL, with a lower limit of quantitation of 1.46 ng/mL. Both intra- and interday precisions were within 11.05%, and the accuracy ranged from -11.5 to 5.51%. The extraction recovery and matrix effect were within acceptable limits. Stability tests showed that PepE remained stable throughout the analytical procedure. The validated method was then used to analyze the pharmacokinetics of PepE administered to rats orally (12.5 and 25 mg/kg) or intravenously (6.25 and 12.5 mg/kg)., (Copyright © 2017 John Wiley & Sons, Ltd.)

The clinical presentation, autopsy findings and toxicology results in an acute fatality involving N-ethylpentylone, a new cathinone derivative, are described. Law enforcement transported a male who was agitated and exhibiting unusual behavior to a local hospital. Upon arrival at the hospital, his body temperature was 105.5 degrees Fahrenheit and his blood pH was 6.7. Clinical laboratory analysis revealed elevated troponins, rhabdomyolysis, hypoglycemia, hepatic and renal injury, respiratory failure and disseminated intravascular coagulation. He was intubated and admitted to the intensive care unit, treated with cooling blankets, bicarbonate and intravenous fluids. Despite medical treatment, he went into cardiac arrest and was pronounced dead ~36 h after admission. Autopsy findings identified some abrasions on his arms and legs, a bloody nose and a mildly enlarged heart. Antemortem blood was analyzed by gas chromatography coupled with a mass spectrometer which identified N-ethylpentylone. Based on clinical presentation, autopsy findings and toxicology results, the medical examiner concluded the cause of death was intoxication by N-ethylpentylone and the manner of death was accident., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

3,4-methylenedioxypyrovalerone (MDPV) is a synthetic cathinone belonging to the class of α-pyrrolidinophenones that become increasingly popular as a designer psychostimulant. Here, we report a comprehensive collection of MDPV exposure with quantitative serum level confirmation in Germany. During the years 2014-2016, we could proof consumption of MDPV in 23 cases where urine and blood samples were submitted to our laboratory by the police of Lower Saxony. Most of the samples underwent systematic toxicological analysis by gas chromatography-mass spectrometry (GC-MS), where MDPV could be detected in urine and/or serum samples. The determined concentrations of MDPV in serum showed a high variability, ranging from traces (<10ng/mL) up to 576ng/mL with a mean concentration of 118ng/mL and median of 47ng/mL. The majority of MDPV users were men (87%) and the age ranged from 23 to 49 years (mean 35.9, median 37 years). For most of the analytically confirmed MDPV cases we could prove co-consumption of other psychotropic drugs with frequent occurrence of opiates and cannabinoids in 22% of the cases, followed by benzodiazepines and cocaine in 17%. Analysis of urine samples by GC-MS disclosed the presence of MDPV and its metabolites 2'-oxo-MDPV, demethylenyl-MDPV, demethylenyl-methyl-MDPV, demethylenyl-oxo-MDPV, demethylenyl-methyl-oxo-MDPV and demethylenyl-methyl-N,N-bisdealkyl-MDPV. The metabolite pattern substantiates previous suggestions for principle metabolic pathways of MDPV in humans., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Recreational use of substituted cathinones continues to be an emerging public health problem in the United States; cathinone derivatives α-pyrrolidinopentiophenone (α-PVP) and 3,4-methylenedioxypyrovalerone (MDPV), which have been linked to human fatalities and show high potential for abuse liability in animal models, are of particular concern. The objective of this study was to develop an immunotherapeutic strategy for attenuating the effects of α-PVP and MDPV in rats, using drug-conjugate vaccines created to generate antibodies with neutralizing capacity. Immunoconjugates (α-PVP-KLH and MDPV-KLH) or the control carrier protein, keyhole limpet hemocyanin (KLH), were administered to groups (N = 12) of male Sprague-Dawley rats on Weeks 0, 2 and 4. Groups were administered α-PVP or MDPV (0.0, 0.25, 0.5, 1.0, 5.0 mg/kg, i.p.) in acute drug challenges and tested for changes in wheel activity. Increased wheel activity produced by α-PVP or MDPV in the controls was attenuated in the α-PVP-KLH and MDPV-KLH vaccinated groups, respectively. Rectal temperature decreases produced by MDPV in the controls were reduced in duration in the MDPV-KLH vaccine group. A separate group (N = 19) was trained to intravenously self-administer α-PVP (0.05, 0.1 mg/kg/inf) and vaccinated with KLH or α-PVP-KLH, post-acquisition. Self-administration in α-PVP-KLH rats was initially higher than in the KLH rats but then significantly decreased following a final vaccine booster, unlike the stable intake of KLH rats. The data demonstrate that active vaccination provides functional protection against the effects of α-PVP and MDPV, in vivo, and recommend additional development of vaccines as potential therapeutics for mitigating the effects of designer cathinone derivatives., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

ORKAMBI (ivacaftor-lumacaftor [LUMA]) and KALYDECO (ivacaftor; IVA) are two new breakthrough cystic fibrosis (CF) drugs that directly modulate the activity and trafficking of the defective CFTR underlying the CF disease state. Currently, no therapeutic drug monitoring assays exist for these very expensive, albeit, important drugs. In this study, for the first time HPLC and LC-MS methods were developed and validated for rapid detection and quantification of IVA and its major metabolites hydroxymethyl-IVA M1 (active) and IVA-carboxylate M6 (inactive); and LUMA in the plasma and sputum of CF patients. With a mobile phase consisting of acetonitrile/water:0.1% formic acid (60:40v/v) at a flow rate of 1mL/min, a linear correlation was observed over a concentration range from 0.01 to 10μg/mL in human plasma (IVA R 2 >0.999, IVA M1 R 2 >0.9961, IVA M6 R 2 >0.9898, LUMA R 2 >0.9954). The assay was successfully utilized to quantify the concentration of LUMA, IVA, M1 and M6 in the plasma and sputum of CF patients undergoing therapy with KALYDECO (IVA 150mg/q12h) or ORKAMBI (200mg/q12h LUMA-125mg/q12h IVA). The KALYDECO patient exhibited an IVA plasma concentration of 0.97μg/mL at 2.5h post dosage. M1 and M6 plasma concentrations were 0.50μg/mL and 0.16μg/mL, respectively. Surprisingly, the ORKAMBI patient displayed very low plasma concentrations of IVA (0.06μg/mL) and M1 (0.07μg/mL). The M6 concentrations (0.15μg/mL) were comparable to those of the KALYDECO patient. However, we observed a relatively high plasma concentration of LUMA (4.42μg/mL). This reliable and novel method offers a simple and sensitive approach for therapeutic drug monitoring of KALYDECO and ORKAMBI in plasma and sputum. The introduction of the assay into the clinical setting will facilitate pharmacokinetics/pharmacodynamic analysis and assist clinicians to develop more cost effective and efficacious dosage regimens for these breakthrough CF drugs., (Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.)

This is a case report of a fatal intoxication in Cyprus related to 3,4-methylenedioxypyrovalerone (MDPV) and 2-(methylamino)-1-phenylpentan-1-one (pentedrone) intake combined with antipsychotic and antidepressant substances. A 42- year old man with a history of serious psychiatric illness was found unresponsive in his bed. Seized materials were also found close to his body. The forensic autopsy reported myocardial infarction due to multidrug intoxication. Toxicology screening in blood and urine was applied. Biological specimens were analysed by enzyme immunoassay procedure and GC/MS. MDPV, pentedrone and etizolam detected and quantitated in blood and urine. Other drugs quantitated in blood were also olanzapine, mirtazapine, and ephedrine. This was the first fatal case reported in Cyprus associated with new psychoactive substances. Additionally, this was the first case reported to Early Warning System of the European Monitoring Center of Drugs and Drug Abuse (EMCDDA), related to multidrug intoxication, attributed to the consumption of cathinones, designer benzodiazepines, and other drugs., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

A sensitive, selective and rapid liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS/MS) method has been developed for the quantification of the novel transforming growth factor-β (TGF-β) inhibitor SB-505124 in rat plasma and then validated. Plasma samples were prepared by simple protein precipitation. Separation was performed on a Diamonsil ODS chromatography column using a mobile phase of acetonitrile and 0.1% (v/v) aqueous formic acid. SB-505124 and the internal standard doxorubicin were detected in the positive ion mode using multiple reaction monitoring of the transitions at m/z 336.2→320.1 and 544.2→397.2, respectively. Calibration curve was linear (r>0.9996) over a concentration range of 10-5000 ng/mL with the lower quantification limit of 10 ng/mL. Both intra- and inter-day precision were within 6.5% and trueness were not more than 3.1%. Extraction recovery and matrix effect were within acceptable limits. Stability tests showed that SB-505124 and the IS remained stable throughout the analytical procedure. The validated LC-MS/MS method was then used to analyze the pharmacokinetics of SB-505124 administered to rats intravenously (8 mg/kg) or orally (10 mg/kg). Oral bioavailability of SB-505124 was calculated as 76.4%, indicating the potential of SB-505124 as an orally administered drug., (Copyright © 2015 Elsevier B.V. All rights reserved.)

3,4-methylenedioxypyrovalerone or MDPV is a synthetic cathinone with psychostimulant properties more potent than cocaine. We quantified this drug in the striatum after subcutaneous administration to rats. MDPV reached the brain around 5 min after its administration and peaked at 20-25 min later. The elimination half-life in the striatum (61 min) correlates with the decrease in the psychostimulant effect after 60 min. Around 11% of the administered dose reached the striatum and, considering a homogeneous brain distribution, we determined that around 86% of the plasma MDPV is distributed to the brain. MDPV induced a dose-dependent increase in locomotor activity, rearing behaviour and stereotypies, all prevented by haloperidol. A plot of locomotor activity or stereotypies versus MDPV striatal concentrations over time showed a direct relationship between factors. No free MDPV metabolites were detected in plasma, at any time, but hydrolysis with glucuronidase allowed us to identify mainly three metabolites, one of them for the first time in rat plasma. The present results contribute to evidence that MDPV induces hyperlocomotion mainly through a dopamine-dependent mechanism. Good correlation between behavioural effects and striatal levels of MDPV leads us to conclude that its psychostimulant effect is mainly due to a striatal distribution of the substance. The present research provides useful information on the pharmacokinetics of MDPV, and can help design new experiments with kinetics data as well as provide a better understanding of the effects of MDPV in humans and its potential interactions., (© The Author(s) 2015.)

Context: In the recent years, there have been an increasing number of new psychoactive substances (NPS) available through marketing and sale on the Internet. The stimulant 3,4-methylenedioxypyrovalerone (MDPV) is a potent dopamine reuptake inhibitor, which can cause serious intoxications requiring intensive care and even fatality. This report from the STRIDA project presents the prevalence, laboratory results, and clinical features in a series of intoxications involving MDPV over a 5-year period., Study Design: Observational case series of consecutive patients with admitted or suspected intake of NPS presented at hospitals in Sweden from 2010 to 2014., Patients and Methods: Blood and/or urine samples were collected from intoxicated patients with admitted or suspected intake of NPS presenting at hospitals over the country. Analysis of NPS was performed by a liquid chromatography-tandem mass spectrometry multicomponent method. Clinical data were collected when caregivers consulted the Swedish Poisons Information Centre and also retrieved from medical records. The severity of poisoning was graded retrospectively using the poisoning severity score., Results: During the 5-year study period, the number of MDPV-related inquiries to the Poisons Information Centre was 662 out of a total ∼4500 suspected NPS-related inquiries (∼15%), and 201 analytically confirmed MDPV intoxications were enrolled in the study. The study period covered the period when the use of MDPV in Sweden was at its peak and also the decline to an almost zero level. The age range of patients was 18-68 (mean 36, median 35) years, and 71% were males. The MDPV concentrations in serum ranged between 1.0 ng/mL and 1509 ng/mL (mean 63.6, median 20) and between 1.0 ng/mL and 81 000 ng/mL (mean 3880, median 1160) in urine. The urinary values were also creatinine corrected for variation in urine dilution, and the MDPV/creatinine ratio ranged between 0.10 ng/mmol and 2480 ng/mmol (mean 247, median 92.6). There was a statistically significant association between the serum MDPV concentration and the urinary MDPV/creatinine ratio, for 118 cases where both data were available (r = 0.764; p < 0.0001, Spearman's rank correlation). In 30 (15%) cases, MDPV was the single psychoactive substance identified in the serum or urine specimens. In the other 171 cases, other psychoactive substances were detected together with MDPV. The additional substances (n = 61) comprised of both conventional drugs of abuse, other NPS (n = 39), pharmaceuticals, and ethanol. The cathinone-derivative alpha-pyrrolidinovalerophenone (α-PVP) was the most frequent other NPS, and was detected in 58 (29%) cases, followed by methylone in 14 (7%) cases. The main clinical manifestations reported in patients testing positive for MDPV included agitation, tachycardia (≥100/min), and hypertension (systolic blood pressure ≥140 mmHg), which were observed in 130 (67%), 106 (56%), and 65 (34%) cases, respectively. Other symptoms included hallucinations (n = 31, 16%), delirium (n = 29, 15%), hyperthermia (>39°C/102.4°F; n = 18, 10%), and rhabdomyolysis (n = 16, 8%). In MDPV intoxications with serum levels >100 ng/mL, the cases were graded as more severe and hyperthermia was less common., Conclusions: In a large number of analytically confirmed MDPV intoxications from mostly polydrug users, the urine and serum MDPV concentrations showed a high variability. The clinical features were consistent with a severe sympathomimetic toxidrome. The results also demonstrated that MDPV prevailed as a drug of abuse for a long time, after its classification as a narcotic substance and despite a high incidence of severe poisonings.

In this study, we sought to determine what impact the banning of 3, 4- methylenedioxypyrovalerone (MDPV) had on the incidence of MDPV-positive findings and on user profiles in driving under the influence of drugs (DUID) and postmortem (PM) investigations in Finland. All MDPV-positive cases and a selection of corresponding court cases between 2009 and 2012 were examined. The median serum concentration of MDPV in DUID cases was 0.030 mg/L and in PM blood 0.12 mg/L. The number of MDPV-positive cases decreased both in DUID and PM investigations after the drug was banned. The decrease in the mean monthly numbers of MDPV-positive DUID cases was 51.1%. In court cases, MDPV was rarely mentioned until banned and frequently mentioned thereafter. Of the convicted, 37% were without a fixed abode, 98% had other charges besides that of DUID, and 13% appeared in the study material more than once. In MDPV-positive PM cases, the proportion of suicides was very high (24%). Research on new psychoactive substances is required not only to support banning decisions but more importantly to be able to provide a scientific assessment of the risks of these new substances to the public and potential users.

Purpose: NSC 743400 is a novel synthetic indenoisoquinoline analog under development as an anticancer agent. It is a potent topoisomerase I inhibitor with potential therapeutic advantages over FDA-approved camptothecin derivatives. In preparation for clinical development of NSC 743400, we determined the pharmacokinetics after administration to rats and dogs., Methods: NSC 743400 was administered intravenously at a dose of 12 or 24 mg/m(2) to rats (single bolus) or 10, 50, 100, 215, 430, or 646 mg/m(2) (intravenous infusion) or 860 or 1720 mg/m(2) (orally) to dogs., Results: Intravenously administered NSC 743400 was eliminated from both species with an estimated t 1/2 of 2-5 h in rat and 6-14 h in dog. Elimination t 1/2 increased with dose in dog. Area under the plasma concentration-versus-time curve (AUC) was comparable in both species, at about 300-400 h ng/mL for the approximately 10 mg/m(2) dose groups. Overall, AUC values increased proportionally with dose for both species but had evidence of more than proportional exposure at the highest doses. Oral dosing resulted in variable drug absorption., Conclusions: The pharmacokinetic data were used to plan first-in-human clinical trials.

CSUOH0901, a novel anticancer derivative of nimesulide, exhibits very promising anticancer activities in various cancer cell lines. In order to support further pharmacological and toxicological studies of this promising anticancer drug candidate, an LC-MS/MS method was developed and validated in accordance with the US Food and Drug Administration guidelines. The drug molecules were extracted from plasma samples by protein precipitation and then analyzed with LC-ESI-MS/MS. An excellent analyte separation was achieved using a phenomenex C18 column with a mobile phase of 90% methanol and 5 m m of ammonium formate. The validated linear dynamic range was between 0.5 and 100 ng/mL and the achieved correlation coefficient (r(2)) was >0.9996. The results of inter- and intra-day precision and accuracy were satisfactory, that is, <12% for accuracy and within ±5% for precision at a low and high quality control concentrations, respectively. In addition, the analyte and internal standard (JCC76) were found to be stable under the storage conditions at -20°C for about 2 months. Hence, the acquired results proved that the LC-ESI-MS/MS method developed is precise, accurate and selective for the quantification of CSUOH0901 in plasma, and can be used for pharmacokinetic studies., (Copyright © 2014 John Wiley & Sons, Ltd.)

Highly electronegative low-density lipoprotein (LDL) L5 induces endothelial cell (EC) apoptosis, which leads to the development of atherosclerosis. We examined the effects of sesamol (1), a natural organic component of sesame oil, on plasma L5 levels and atherosclerosis development in a rodent model and on the L5-induced apoptosis of ECs. Syrian hamsters, which have an LDL profile similar to that of humans, were fed a normal chow diet (control), a high-fat diet (HFD), or a HFD supplemented with the administration of 50 or 100 mg/kg of 1 via oral gavage (HFD+1) for 16 weeks (n = 8 per group). Hamsters in the HFD+1 groups had reduced plasma L5 levels when compared with the HFD group. Oil Red O staining showed that atherosclerotic lesion size was markedly reduced in the aortic arch of hamsters in the HFD+1 groups when compared with that in the HFD group. In human aortic ECs, 0.3-3 μM 1 blocked L5-induced apoptosis in a dose-dependent manner. Further mechanistic studies showed that 1 inhibited the L5-induced lectin-like oxidized LDL receptor-1 (LOX-1)-dependent phosphorylation of p38 MAPK and activation of caspase-3 and increased phosphorylation of eNOS and Akt. Our findings suggest that sesamol (1) protects against atherosclerosis by reducing L5-induced atherogenicity.

Previous research has shown that resveratrol can increase cerebral blood flow (CBF) in the absence of improved cognitive performance in healthy, young human subjects during the performance of cognitively demanding tasks. This lack of cognitive effects may be due to low bioavailability and, in turn, reduced bioefficacy of resveratrol in vivo. Piperine can alter polyphenol pharmacokinetics, but previous studies have not investigated whether this affects the efficacy of the target compound. Therefore, the objective of the present study was to ascertain whether co-supplementation of piperine with resveratrol affects the bioavailability and efficacy of resveratrol with regard to cognition and CBF. The present study utilised a randomised, double-blind, placebo-controlled, within-subjects design, where twenty-three adults were given placebo, trans-resveratrol (250 mg) and trans-resveratrol with 20 mg piperine on separate days at least a week apart. After a 40 min rest/absorption period, the participants performed a selection of cognitive tasks and CBF was assessed throughout the period, in the frontal cortex, using near-IR spectroscopy. The presence of resveratrol and its conjugates in the plasma was confirmed by liquid chromatography-MS analysis carried out following the administration of the same doses in a separate cohort (n 6). The results indicated that when co-supplemented, piperine and resveratrol significantly augmented CBF during task performance in comparison with placebo and resveratrol alone. Cognitive function, mood and blood pressure were not affected. The plasma concentrations of resveratrol and its metabolites were not significantly different between the treatments, which indicates that co-supplementation of piperine with resveratrol enhances the bioefficacy of resveratrol with regard to CBF effects, but not cognitive performance, and does this without altering bioavailability.

Synthetic cathinones are recreational drugs that mimic the effects of illicit stimulants like cocaine, amphetamine or Ecstasy. Among the available synthetic cathinones in the United States, 3,4-methylenedioxypyrovalerone (MDPV) is commonly abused and associated with dangerous side effects. MDPV is a dopamine transporter blocker 10-fold more potent than cocaine as a locomotor stimulant in rats. Previous in vitro and in vivo studies examining MDPV metabolism reported 3,4-dihydroxypyrovalerone (3,4-catechol-PV) and 4-hydroxy-3-methoxypyrovalerone (4-OH-3-MeO-PV) as the two primary metabolites. We developed and validated a liquid chromatography-high resolution mass spectrometry method to quantify MDPV and its primary metabolites in 100 μL human and rat plasma. Plasma hydrolysis was followed by protein precipitation before analysis. Limits of detection were 0.1 μg L(-1), with linear ranges from 0.25 to 1000 μg L(-1). Process efficiency, matrix effect, total imprecision (%CV) and accuracy (%target) were 36-93%, from -8 to 12%, 2.1 to 7.3% and 86 to 109%, respectively. MDPV and metabolites were stable at room temperature for 24 h, 4 °C for 72 h and after 3 freeze-thaw cycles with less than 10% variability. Human-rat plasma cross validation demonstrated that rat plasma could be accurately quantified against a human plasma calibration curve. As proof of this method, rat plasma specimens were analyzed after intraperitoneal and subcutaneous dosing with MDPV (0.5 mg kg(-1)). MDPV, 3,4-catechol-PV and 4-OH-3-MeO-PV concentrations ranged from not detected to 107.5 μg L(-1) prior to and up to 8h after dosing. This method provides a simultaneous quantification of MDPV and two metabolites in plasma with good selectivity and sensitivity., (Published by Elsevier B.V.)

In our previous study, piperlonguminine from the fruit of Piper chaba was reported to promote adipogenesis in 3T3-L1 cells like the peroxisome proliferator-activated receptor-γ (PPARγ) agonist, troglitazone. In the present study, the mode of action of piperlonguminine in cells was examined. Piperlonguminine increased mRNA levels of adiponectin, glucose transporter 4, and fatty acid-binding protein (aP2). It also increased mRNA levels of PPARγ2 but, unlike troglitazone, piperlonguminine did not activate PPARγ directly in a nuclear receptor cofactor assay. Analyses of plasma from mice treated with piperlonguminine, piperine, and retrofractamide A, and an extract of the fruit, showed that concentrations of piperlonguminine were higher than those of piperine and retrofractamide A, and that the "area-under-the-curve" of piperine increased following in vivo administration of the extract.

Cathinone derivatives (bath salts) have emerged as the latest drugs of abuse. 3,4-methylenedioxypyrovalerone (MDPV) is the primary active ingredient in bath salts used in this country. This article presents the second reported cause of death by MDPV intoxication alone. In April 2011, a delusional man was emergently brought to a hospital, where he self-reported bath salt usage. He became agitated, developed ventricular tachycardia, hyperthermia, and died. Comprehensive alcohol and drug testing was performed. Using the alkaline drug screen, heart blood contained 0.7 mg/L MDPV and peripheral blood contained 1.0 mg/L MDPV. His bizarre behavior with life-threatening hyperthermia was consistent with an MDPV-induced excited delirium state. MDPV is not yet found by routine immunoassay toxicology screens. Testing for MDPV should be considered in cases with a history of polysubstance abuse with stimulant type drugs, report of acute onset of psychogenic symptoms, excited delirium syndrome, or presentation in a hyperthermic state., (© 2013 American Academy of Forensic Sciences.)

3,4-Methylenedioxypyrovalerone (MDPV) is a cathinone derivative. It has recently been classified as a controlled substance in many countries. This substance is a stimulant that can be snorted, smoked or taken orally. MDPV has been determined in biological material from four cases sent to the Institute of Forensic Research in 2011. In the first case, a passenger car crashed into a truck; the driver of the vehicle suffered severe injuries, resulting in his death. In the second case, biological material was obtained from the decedent male individual, who did not wake up after a party. In the two cases, the material was secured on suspicion of the possession of narcotic drugs or psychotropic substances, in which the suspects admitted to using "legal highs." The MDPV blood concentrations of the deceased driver and deceased man were 38 and 17 ng/mL, respectively. In the two other cases, the determined concentrations were 306 and 124 ng/mL. However, MDPV was not the sole substance detected in these cases: in each, other drugs were also determined. Analyses of blood were conducted using liquid chromatography-tandem mass spectrometry.

3,4-Methylenedioxypyrovalerone (MDPV) is a psychoactive, synthetic analog of the central nervous system stimulant cathinone. Its recent popularity as a recreational drug in the United States has led to numerous reports to poison control centers across the country. As with other synthetic cathinones, the recreational use of MDPV has resulted in death. MDPV is thought to exert its pharmacologic effects by inhibiting the reuptake of dopamine and norepinephrine. This report describes the case of an exposure of a 39-year-old male to MDPV, which resulted in his death. Postmortem concentrations of MDPV in various tissues were measured. The detection of MDPV in tissues and fluids was accomplished using gas chromatography-mass spectrometry analysis after solid-phase extraction. Blood analysis also demonstrated therapeutic levels of lamotrigine, fluoxetine, risperidone, benztropine, pseudoephedrine and ibuprofen. The detection of cathinones in hair was conducted using high-performance liquid chromatography-tandem mass spectrometry after solid-phase extraction. MDPV was uniformly distributed among multiple tissues (blood, brain, muscle, cerebrospinal fluid and lung) at concentrations of approximately 0.4 to 0.6 µg/mL. Tissue and fluids responsible for detoxification/excretion had higher concentrations of MDPV (kidney, liver and bile > 0.8 µg/mL). A blood concentration ≥ 0.4 µg/mL was judged sufficient to cause death. The cause of death was ruled MDPV intoxication resulting in cardiac arrhythmia.

The analysis of designer drugs, including those in the synthetic cathinone and piperazine classes, may be complicated by the poor stability of these compounds in biological specimens. The stability of four of these compounds was investigated: 3,4-methylenedioxypyrovalerone, 4-methyl-N-methylcathinone (mephedrone), N-benzylpiperazine and 1-[3-(trifluoromethyl)phenyl]piperazine. Compound stability was monitored in three different biological matrices when each matrix was stored under three different conditions. These matrices and conditions included human whole blood, human serum and human urine, each stored at -20, 4 and 22°C for a period of 14 days in the dark in a sealed glass container. Analysis by liquid chromatography-tandem mass spectrometry was performed on Day 1 to establish the initial concentration for each drug in each specimen type, and then the samples were divided into three parts for storage under the various conditions. Analysis was performed in triplicate on Days 2, 4, 7 and 14 for each specimen type under each storage condition and the results were compared to those obtained on Day 1. Following analysis of the data, it became clear that mephedrone was not stable, and that care must be taken following specimen receipt to ensure minimal degradation.

Introduction: The use of designer drugs commonly marketed as bath salts or plant food has risen dramatically in recent years. Several different synthetic cathinones have been indentified in these products, including mephedrone, 3,4-methylenedioxypyrovalerone (MDPV), and 4-fluoromethcathinone (flephedrone). We report a case of bath salt intoxication with quantitative MDPV and flephedrone levels in a patient's serum and urine, and from the bath salt product., Case Report: A 23-year-old male with a prior psychiatric history arrived via EMS for bizarre behavior, suicidality, and hallucinations after reportedly insufflating a bath salt. He was found to have MDPV levels of 186 and 136 ng/mL in his serum and urine, respectively, and flephedrone levels of 346 and 257 ng/mL in the serum and urine, respectively. The white powder in question was found to contain 143 μg MDPV and 142 μg flephedrone per milligram powder. His psychosis and agitation resolved with lorazepam, droperidol, and observation in the emergency department., Discussion: Agitation, psychosis, movement disorders, tachycardia, and hypertension have all been attributed to the use of MDPV; there are no prior reports detailing clinical experience with flephedrone. Considering that our patient's serum flephedrone levels were twofold higher than his MDPV level, it is likely flephedrone contributed to his clinical toxicity. This case suggests the possibility that fluorinated cathinones, such as flephedrone, may have altered metabolism and/or elimination which may affect their course of clinical toxicity. This case highlights the evolving composition of synthetic cathinones found in bath salt products.

Drugs derived from amphetamine, methamphetamine and their methylenedioxy- analogues, although being sold as plant food or bath salts, are being used as legal alternatives to scheduled amphetamine stimulants. These products often contain methylone, mephedrone and methylenedioxypyrovalerone (MDPV)--three amphetamine derivatives shown to have strong pharmacological effects. Four postmortem cases were analyzed for methylone, mephedrone and MDPV, with drug levels quantitated in multiple biological matrices. All four cases had detectable levels of methylone, with heart blood concentrations of 0.740, 0.118, 0.060 and 1.12 mg/L. Analysis of several tissue samples shows that methylone does not sequester in a particular tissue type after death. The average liver-to-blood ratio was 2.68. Two cases also had MDPV present, but insufficient data were collected to formulate a hypothesis on postmortem sequestration or redistribution. Two different extraction methods, as well as analysis of derivatized and underivatized methylone, show that the drug is suitable for analysis in either method. The cases are believed to show one instance of chronic methylone use, with a urine concentration of 38 mg/L.

3,4-Methylenedioxypyrovalerone (MDPV) is a psychoactive drug with potent stimulant properties and potential for abuse and drug dependency. MDPV was recently classified as a Class I drug by Racing Commissioners International, indicating that it is a banned substance in equine athletes because it lacks therapeutic value in horses. To enforce this ban, a sensitive and fast liquid chromatography-tandem mass spectrometry method was needed. It is for this reason that this method was developed for quantification and confirmation of MDPV in equine plasma. Sample preparation involved liquid-liquid extraction. The analyte was analyzed by a triple-quadrupole linear ion trap mass spectrometer in positive multiple-reaction-monitoring and enhanced product ion scan modes. The method was validated for precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), linearity, stability, extraction recovery, matrix effect, dilution accuracy and selectivity. The extraction recovery was >90%. The linearity range was from 5 to 15,000 pg/mL. LOD and LOQ were 2 and 5 pg/mL, respectively. Intra-day and inter-day accuracies were nearly 100%. The method is suitable for screening, quantification and confirmation of MDPV in equine plasma and has been successfully used to detect and confirm the presence of MDPV in equine plasma obtained post-competition.

The alkaloids from Piper longum L. showed protective effects on Parkinson's disease models in our previous study and piperine and piperlonguminine were the two main constituents in the alkaloids. The present study aimed at developing a rapid, sensitive, and accurate UFLC-ESI-MS/MS method and validating it for the simultaneous determination of piperine and piperlonguminine in rat plasma using terfenadine as the internal standard. The analytes and internal standard (IS) were extracted from rat plasma using a simple protein precipitation by adding methanol/acetonitrile (1:1, v/v). A Phenomenex Gemini 3 u C18 column (20 mm × 2.00 mm, 3 μm) was used to separate the analytes and IS using a gradient mode system with a mobile phase consisting of water with 0.1% formic acid (mobile phase A) and acetonitrile with 0.1% formic acid (mobile phase B) at a flow rate of 0.4 mL/min and an operating column temperature of 25°C. The total analytical run time was 4 min. The detection was performed using the positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode with transitions at m/z 286.1-201.1 for piperine, m/z 274.0-201.1 for piperlonguminine, and m/z 472.4-436.4 for the IS. The calibration curves were both linear (r>0.995) over a concentration range of 1.0 to 1000 ng/mL; the lower limit of quantification (LLOQ) was 1.0 ng/mL for both piperine and piperlonguminine. The intra-day and inter-day precisions (RSD %) were <12.1%, accuracies ranged from 86.6 to 120%, and recoveries ranged from 90.4 to 108%. The analytes were proven stable in the short-term, long-term, and after three freeze-thaw cycles. The method was successfully applied to pharmacokinetic studies of piperine and piperlonguminine in rats after oral administration of alkaloids from P. longum L., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A rapid, selective, and sensitive liquid chromatography-atmospheric pressure chemical ionization (APCI) tandem mass spectrometry method was developed for the simultaneous determination of dimethoxyaschantin, dimethylliroresinol, dimethylpinoresinol, epimagnolin A, fargesin and magnolin, the pharmacologically active ingredients of Magnolia fargesii in rat plasma. These tetrahydrofurofuranoid lignans were extracted from rat plasma using tert-butyl methyl ether at pH 7.4. The analytes were separated on a Pinnacle DB biphenyl column with 65% methanol in 10 mm ammonium formate (pH 3.0) and detected by APCI tandem mass spectrometry in the selective reaction monitoring mode. The calibration curves were linear (r(2) ≥ 0.996) over the concentration range of 20.0-1000 ng/mL for six tetrahydrofurofuranoid lignans. The lower limit of quantification for these lignans was 20.0 ng/mL with 50 µL of plasma sample. The intra- and inter-assay coefficient of variation and relative error for the six tetrahydrofurofuranoid lignans at four quality control concentrations were 0.2-9.9% and -8.5-8.2%, respectively. There was no matrix effect for the six tetrahydrofurofuranoid lignans and tolterodine (internal standard). The pharmacokinetics of dimethylliroresinol, dimethylpinoresinol, epimagnolin A, fargesin and magnolin were evaluated after oral administration of a purified extract isolated from dried flower buds of Magnolia fargesii at doses of 5.5, 11.0 and 22.0 mg/kg in male rats., (Copyright © 2010 John Wiley & Sons, Ltd.)

Starting in 2008 a new designer drug, 3,4-methylenedioxypyrovalerone (MDPV) appeared among users of illegal drugs in Finland. Since then there have been several seizures of MDPV by police and customs and it has been connected to many crimes of different types. In this study the incidence and impact of the use of MDPV in drivers suspected of being under the influence of drugs (DUID) in Finland was assessed. Since autumn 2009, blood samples from drivers suspected of DUID in Finland have been analysed for the presence of MDPV. A new LC-MS/MS method for the determination of MDPV in serum was established. In order to assess the impact of MDPV on driving performance, drug and alcohol findings of positive MDPV cases were compared with data from the clinical examination carried out while the suspect was under arrest. In a period of one year there were 259 positive MDPV cases from apprehended drivers (5.7% of all confirmed DUID cases). In 80% of the cases in which MDPV was found, amphetamine was also present. Benzodiazepines were also frequently found together with MDPV, which was to be expected since in Finland, in our experience, stimulants are very often used together with benzodiazepines. In most cases it remained unclear whether the observed psycho-physical achievement deficiency was induced by MDPV because the concentrations of other drugs, especially other stimulants, were often high. However, in some subjects, MDPV, or MDPV in combination with other substances was the most probable cause of the impairment. The concentrations of MDPV varied from 0.016mg/L to over 8.000mg/L. Little is known about the pharmacology of MDPV. However, based on our findings it is clear that MDPV has a serious impact on traffic safety in Finland., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Ethyl piperate is an effective lipid-lowering drug candidate synthesized from piperine. However, its pharmacokinetic characteristics and oral absorption process remain unclear. A liquid chromatography-tandem mass spectrometry method was applied to determine the oral bioavailability of ethyl piperate. Simulated gastrointestinal pH conditions and intestinal washings were prepared to investigate their contributions to the loss of ethyl piperate. Hydrolysis by carboxylesterase (CES) was evaluated in vitro using microsomes and S9 fractions. In situ intestinal single-pass perfusion experiments were performed to estimate the role of CES in ethyl piperate absorption. The bioavailability of ethyl piperate was extremely low (0.47%) in hamster independent of gastrointestinal environmental effects. Ethyl piperate was a typical substrate of CES with kinetic parameters K(m) and V(max) of 7.56 ± 1.491 μM and 0.16 ± 0.008 nmol · min(-1) · mg protein(-1), respectively. CES was responsible for 85.8% of the intestinal hydrolysis of ethyl piperate. Specific inhibition of CES with bis-p-nitrophenyl phosphate (BNPP), decreased degradation clearance to 36% of control with no significant change in absorption clearance. This contrasted with the results of Caco-2 monolayer experiments, which showed a dramatic increase in the apparent permeability coefficient after BNPP treatment. mRNA levels for the CES isozyme, CES2A3, were similar among the three regions of hamster intestine and 60% less than those in liver; CES1B1 mRNA levels were even lower in the intestine and showed a proximal-to-distal decrease. In conclusion, CES markedly contributes to intestinal first-pass hydrolysis of ethyl piperate that is sufficient, but not necessary, to cause the observed extremely low bioavailability.