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Three plasticizers, namely bis (3,5,5-trimethylhexyl) phosphate (TMHPh), di(propylene glycol) dibenzoate (DiPGDB), and tri-n-butyl trimellitate (TBTM), were recently identified and reported in high concentrations in indoor dust from Belgian homes. In this study, their behavior within the human body was investigated by generating Phase I biotransformation products for the first time. Human liver microsomes (HLMs) were used following an in vitro assay and liquid chromatography time of flight mass spectrometry (LC-QTOF-MS) was employed for the analysis. Biotransformation products were identified for TMHPh as products of hydroxylation reactions that took place in one or two positions in the structure of the substrate. For DiPGDB, biotransformation products were formed after hydrolysis of carboxylic esters and oxidative-O-dealkylation. For TBTM, biotransformation products were formed through hydrolysis of the different carboxylic esters of the molecule, in agreement with studies on structurally similar compounds. The generated results can contribute to biomonitoring studies creating new knowledge on human exposure to emerging compounds and on the metabolism of xenobiotics.

Alcohol abuse has become a serious health issue worldwide. Ketamine can reduce addiction risk among patients with alcohol use disorders. This study aimed to determine the effects of alcohol on the pharmacokinetics of ketamine during long-term alcohol exposure.An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for determination of ketamine and norketamine was developed and validated. A total of 15 rats were given 40% alcohol for 3 weeks. The pharmacokinetics of ketamine were measured at time zero, 1 week, 2 weeks, and 3 weeks after alcohol exposure. The metabolic capability of liver CYP450 was evaluated using three probe drugs: metoprolol, phenacetin, and tolbutamide.During drinking of 40% alcohol, the AUC(0-t), AUC(0-∞), and Cmax of ketamine and norketamine significantly increased, while V and CL significantly decreased with time (p 0.001). The pharmacokinetic changes of norketamine were highly consistent with ketamine. Additionally, the concentration ratio of norketamine/ketamine in sample time also decreased over time. However, there were no pharmacokinetic changes of three probe drugs, which indicated there was no significant change of liver CYPs activities.Alcohol significantly increases plasma concentration of ketamine and norketamine. The effect of alcohol on pharmacokinetics of ketamine should be considered in clinical therapy.

In this research, the gravimetric method was utilized to measure the solubility of phenacetin from 283.15 to 323.15 K under atmospheric pressure in 12 solvents, including methanol, ethanol, 1-propa...

Paeoniflorin is the major constituent in extracts of the paeony root, the purpose of the present study was to assess the effects of paeoniflorin on the activities and mRNA expression of the rat hepatic drug-metabolizing enzymes cytochrome P450 (CYP1A2), CYP2C11 and CYP3A1

Hydrolytic reactions constitute an important pathway of drug metabolism and a significant route of prodrug activation. Many ophthalmic drugs and prodrugs contain ester groups that greatly enhance their permeation across several hydrophobic barriers in the eye before the drugs are either metabolized or released, respectively, via hydrolysis. Thus, the development of ophthalmic drug therapy requires the thorough profiling of substrate specificities, activities, and expression levels of ocular esterases. However, such information is scant in the literature, especially for preclinical species often used in ophthalmology such as rabbits and pigs. Therefore, our aim was to generate systematic information on the activity and expression of carboxylesterases (CESs) and arylacetamide deacetylase (AADAC) in seven ocular tissue homogenates from these two species. The hydrolytic activities were measured using a generic esterase substrate (4-nitrophenyl acetate) and, in the absence of validated substrates for rabbit and pig enzymes, with selective substrates established for human CES1, CES2, and AADAC (D-luciferin methyl ester, fluorescein diacetate, procaine, and phenacetin). Kinetics and inhibition studies were conducted using these substrates and, again due to a lack of validated rabbit and pig CES inhibitors, with known inhibitors for the human enzymes. Protein expression levels were measured using quantitative targeted proteomics. Rabbit ocular tissues showed significant variability in the expression of CES1 (higher in cornea, lower in conjunctiva) and CES2 (higher in conjunctiva, lower in cornea) and a poor correlation of CES expression with hydrolytic activities. In contrast, pig tissues appear to express only CES1, and CES3 and AADAC seem to be either low or absent, respectively, in both species. The current study revealed remarkable species and tissue differences in ocular hydrolytic enzymes that can be taken into account in the design of esterase-dependent prodrugs and drug conjugates, the evaluation of ocular effects of systemic drugs, and in translational and toxicity studies.

Human arylacetamide deacetylase (AADAC) plays a role in the detoxification or activation of drugs and is sometimes involved in the incidence of toxicity by catalyzing hydrolysis reactions. AADAC prefers compounds with relatively small acyl groups, such as acetyl groups. Eslicarbazepine acetate, an antiepileptic drug, is a prodrug rapidly hydrolyzed to eslicarbazepine. We sought to clarify whether AADAC might be responsible for the hydrolysis of eslicarbazepine acetate. Eslicarbazepine acetate was efficiently hydrolyzed by human intestinal and liver microsomes and recombinant human AADAC. The hydrolase activities in human intestinal and liver microsomes were inhibited by epigallocatechin gallate, a specific inhibitor of AADAC, by 82% and 88% of the control, respectively. The hydrolase activities in liver microsomes from 25 human livers were significantly correlated (r = 0.87, P < 0.001) with AADAC protein levels, suggesting that the enzyme AADAC is responsible for the hydrolysis of eslicarbazepine acetate. The effects of genetic polymorphisms of AADAC on eslicarbazepine acetate hydrolysis were examined by using the constructed recombinant AADAC variants with T74A, V172I, R248S, V281I, N366K, or X400Q. AADAC variants with R248S or X400Q showed lower activity than wild type (5% or 21%, respectively), whereas those with V172I showed higher activity than wild type (174%). Similar tendencies were observed in the other four substrates of AADAC; that is, p-nitrophenyl acetate, ketoconazole, phenacetin, and rifampicin. Collectively, we found that eslicarbazepine acetate is specifically and efficiently hydrolyzed by human AADAC, and several AADAC polymorphic alleles would be a factor affecting the enzyme activity and drug response. SIGNIFICANCE STATEMENT: This is the first study to clarify that arylacetamide deacetylase (AADAC) is responsible for the activation of eslicarbazepine acetate, an antiepileptic prodrug, to eslicarbazepine, an active form, in the human liver and intestines. In addition, we found that several AADAC polymorphic alleles would be a factor affecting the enzyme activity and drug response.

CYP1A2 is one of the main Cytochrome P450 enzymes in the human liver associated with the metabolism of several xenobiotics. CYP1A2 is especially involved in the metabolic activation of different procarcinogens. Therefore, the development of cancer may be inhibited by inhibiting CYP1A2 activity. Here, the inhibitory effect of HYIpro-3-1 and its derivatives on CYP1A2 activity in human liver microsomes (HLM) was studied through LC-MS/MS using a cocktail assay. Among the four compounds, HYIpro-3-1 showed the most selective and strongest inhibitory effect on CYP1A2 at IC50 values of 0.1 µM in HLMs and inhibition was confirmed using purified human CYP1A2. It was determined that inhibition is reversible because the inhibitory effect of HYIpro-3-1 is not dependent on preincubation time. HYIpro-3-1 showed a typical pattern of competitive inhibition for CYP1A2-catalyzed phenacetin O-deethylation, based on the Lineweaver-Burk plot, with a Ki value of 0.05 μM in HLMs; the secondary plot also showed a linear pattern. In our study, HYIpro-3-1 was proposed as a novel inhibitor with the capacity to selectively inhibit CYP1A activity in HLMs. This article is protected by copyright. All rights reserved.

Microcompartments in the form of water-in-oil droplets have been utilized to construct artificial cells and simulate human body environment. However, the performance of subcellular structure involved metabolism in emulsion droplets has not been explored, and the underlying mechanism is still being elucidated. In this work, drug metabolism is presented on the basis of great amounts of microcompartments formed of picoliter-volume droplets with different radius (R), using a commercial four-way valve as a droplet generator. A model substrate, phenacetin, and its metabolite, paracetamol, are quantitatively analyzed by liquid-chromatography (LC) tandem mass spectrometry (MS/MS), and the reaction kinetics is characterized. In microdroplets of varying size (R = 18, 27, 42, and 51 μm, respectively), both conversion ratio and reaction rate constant of the metabolism are influenced in different degree. For instance, the substrate conversion ratio after 60 min of incubation in R = 27 μm droplets improves from 15% to 42%, and the reaction rate constant improves nearly five-fold, compared to that in bulk phase. The influence of microcompartment size on metabolism rate is further explored by simulation using a diffusion-reaction model. The droplet-based strategy is rapid, accurate and cost-efficient, fitting especially into biomimetic metabolism studies.

In the recent work, we have used the density functional theory (DFT) and the molecular dynamics (MD) simulation methods to investigate the adsorption behavior of paracetamol and phenacetin molecules on the boron nitride nanotubes (BNNTs) in the aqueous phase. In this research, several armchair (n,n) BNNTs, where n = 5, 6, and 7 have been used. Two different interactions, including interaction of paracetamol and phenacetin molecules with outer and inner surfaces of BNNTs were studied. The results show that both paracetamol and phenacetin molecules are not adsorbed inside of (5,5) BNNT and are desorbed. It is found that the most stable adsorption corresponds to (6,6) BNNT, when the drug molecule is located inside of the nanotube. The strongest interaction corresponds to adsorption of phenacetin molecule inside of (6,6) BNNT.

Novel HepG2 cell clones 1A2 C2 and 1A2 C7 were independently generated by lentiviral transduction to functionally overexpress cytochrome P450 1A2 (CYP1A2). We found similar and stable CYP1A2 transcript and protein levels in both cell clones leading to specific enzyme activities of about 370 pmol paracetamol x min-1 x mg-1 protein analyzed by phenacetin conversion. Both clones showed dramatically increased sensitivity to the hepatotoxic compound aflatoxin B1 (EC50 < 100 nM) when compared to parental HepG2 cells (EC50 ∼5 mM). Thus, newly established cell lines are an appropriate tool to study metabolism and toxicity of substances depending on conversion by CYP1A2.

Quality is the most important issue in the pharmaceutical field due to the presence of a drug which is considered as safe and therapeutically active agent. In-vitro evaluation ensures their quality, bioavailability as well as optimum therapeutic activity. Paracetamol (acetaminophen) which are the active metabolites of phenacetin is commonly used for the relief of headaches and pains, and is a major ingredient in numerous cold and flu remedies. Paracetamols are available in different brands in Indian market. The main objective of the present study was to conduct the comparative in-vitro dissolution studies of various brands collected from the local market to determine whether all the formulations used were equivalent or significantly different. The calibration curve was constructed covering the concentration range of 1 to 10 mcg/ml at 268 nm by UV spectrophotometer (UV 2203 Double beam spectrophotometer, Shimadzu). Five different brands of Paracetamol of 500 mg conventional tablets from different manufacturers were selected in the study and dissolution testing in Phosphate buffer at pH 7.4 was conducted from each brands for 90 mins by using dissolution testing apparatus USP type-II. The dissolution rate was subjected to various mathematical models like zero order, first order, Higuchi and Hixson-Crowell equations to elucidate the kinetic behavior of drug release from the test samples. Different release kinetics model of all the selected brands was assuring the quality standard of manufacturing. Keywords: Paracetamol, Marketed Tablet, In-Vitro dissolution study, Release profile.

Background:There has been a lack of information about the inhibition of bovine medicines on bovine hepatic CYP450 at their commercial doses and dosing routes.Objective:The aim of this work was to assess the inhibition of 43 bovine medicines on bovine hepatic CYP450 using a combination of in vitro assay and Cmax values from pharmacokinetic studies with their commercial doses and dosing routes in the literature.Methods:Those drugs were first evaluated through a single point inhibitory assay at 3 μM in bovine liver microsomes for six specific CYP450 metabolisms, phenacetin o-deethylation, coumarin 7- hydroxylation, tolbutamide 4-hydroxylation, bufuralol 1-hydroxylation, chlorzoxazone 6-hydroxylation and midazolam 1’-hydroxylation. When the inhibition was greater than 20% in the assay, IC50 values were then determined. The potential in vivo bovine hepatic CYP450 inhibition by those drugs was assessed using a combination of the IC50 values and in vivo Cmax values from pharmacokinetic studies at their commercial doses and administration routes in the literature.Results:Fifteen bovine medicines or metabolites showed in vitro inhibition on one or more bovine hepatic CYP450 metabolisms with different IC50 values. Desfuroylceftiour (active metabolite of ceftiofur), nitroxinil and flunixin have the potential to inhibit one of the bovine hepatic CYP450 isoforms in vivo at their commercial doses and administration routes. The rest of the bovine medicines had low risks of in vivo bovine hepatic CYP450 inhibition.Conclusion:This combination of in vitro assay and in vivo Cmax data provides a good approach to assess the inhibition of bovine medicines on bovine hepatic CYP450.

Background Calycosin (CAL), a type of O-methylated isoflavone extracted from the herb Astralagusmembranaceus (AM), is a bioactive chemical with antioxidative, antiphlogistic and antineoplastic activities commonly used in traditional alternative Chinese medicine. AM has been shown to confer health benefits as an adjuvant in the treatment of a variety of diseases. Aim The main objective of this study was to determine whether CAL influences the cytochrome P450 (CYP450) system involved in drug metabolism. Methods Midazolam, tolbutamide, omeprazole, metoprolol and phenacetin were selected as probe drugs. Rats were randomly divided into three groups, specifically, 5% Carboxymethyl cellulose (CMC) for 8 days (Control), 5% CMC for 7 days + CAL for 1 day (single CAL) and CAL for 8 days (conc CAL), and metabolism of the five probe drugs evaluated using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Results No significant differences were observed for omeprazole and midazolam, compared to the control group. T max and t1/2 values of only one probe drug, phenacetin, in the conc CAL group were significantly different from those of the control group (T max h: 0.50±0.00 vs 0.23±0.15; control vs conc CAL). C max of tolbutamide was decreased about two-fold in the conc CAL treatment group (conc vs control: 219.48 vs 429.56, P Conclusion Calycosin inhibits the catalytic activities of CYP1A2, CYP2D6 and CYP2C9. Accordingly, we recommend caution, particularly when combining CAL as a modality therapy with drugs metabolized by CYP1A2, CYP2D6 and CYP2C9, to reduce the potential risks of drug accumulation or ineffective treatment.

Selenium is an essential nutrient in diets; however, the effects of selenium on enzyme metabolic activation are not currently clear. Cytochromes P450 (CYP450) are major phase I metabolic enzymes involved in the biotransformation of xenobiotics and endogenous compounds to form electrophilic reactive metabolites. To investigate the effect of selenium on CYP450 isoform activity, the Landrace pigs were divided into three groups: the control group (containing Se 0.15 mg/kg), the Se-deficient group (Se 0.03 mg/kg), and the Se-supply group (Se 0.35 mg/kg). After 1 week of administration, a mixed solution (20 mg/kg of dextromethorphan, phenacetin, chlorzoxazone, and 10 mg/kg of testosterone in a CMC-Na solution) was intravenously injected into all pigs. The mixed solution content and pharmacokinetic parameters were assayed by HPLC and DAS, respectively. To investigate the effect of selenium on CYP450 isoform expression, RNA-Seq analysis, Western boltting, and qPCR were used. Results showed that Se-supply group significantly increased the activity and expression of CYP1A2 and CYP2D25, and decreased CYP3A29. Se-deficient group decreased the activity of CYP1A2, CYP2D25, and CYP2E1. These results demonstrated that selenium content affecting the activity or expression of the CYP450 isoform may lead to a food-drug interaction.

A new method of data interpretation based on classical nucleation theory is proposed in this work to elucidate the influence of solvents on the pre-exponential nucleation factor and interfacial energy using the induction time data for three crystallization systems, including isonicotinamide, lovastatin, and phenacetin. In this method, the pre-exponential nucleation factor is replaced by the intrinsic nucleation factor multiplied by temperature and divided by solution viscosity. The proposed method is applied to study the nucleation kinetics of isonicotinamide, lovastatin, and phenacetin among various solvents using the induction time data measured in this work. The results indicate that the intrinsic nucleation factor increases linearly with increasing square root of interfacial energy in various solvents for each system.

Pirfenidone is a first-line drug for the treatment of idiopathic pulmonary fibrosis. The primary metabolic pathways of pirfenidone in humans are 5-hydroxylation and subsequent oxidation to 5-carboxylpirfenidone. The aims of this study were to determine the cytochrome P450 isoforms responsible for pirfenidone 5-hydroxylation and to evaluate their contributions in human liver microsomes (HLM).Among the recombinant P450 isoforms, CYP1A2, CYP2D6, CYP2C19, CYP2A6, and CYP2B6 were shown to catalyse the 5-hydroxylation of pirfenidone. Pirfenidone 5-hydroxylase activity by HLM was inhibited by α-naphthoflavone (by 45%), 8-methoxypsolaren (by 84%), tranylcypromine (by 53%), and quinidine (by 15%), which are CYP1A2, CYP1A2/CYP2A6/CYP2C19, CYP2A6/CYP2C19, and CYP2D6 inhibitors, respectively.In 17 individual HLM donors, pirfenidone 5-hydroxylase activity was significantly correlated with phenacetin O-deethylase (r = 0.89, P

We have developed a sensitive and rapid gold nanoparticle-based immunochromatographic strip (GNP-ICS) for the detection of phenacetin (PNCT) and paracetamol (PAP) using an anti-PNCT monoclonal antibody (mAb). The sensitive anti-PNCT mAb (2D6) had a half maximal inhibitory concentration (IC50) and limit of detection (LOD) of 3.51 and 0.21 ng mL-1, respectively. Additionally, its cross-reactivity with PAP was approximately 10.1%. The developed GNP-ICS assay based on GNP-labeled mAb was sensitive for the detection of PNCT with vLOD and cut-off values of 2.5 and 50 ng mL-1 respectively and a vLOD value of 25 ng mL-1 for PAP. Furthermore, the developed icELISA and GNP-ICS assays were applied to determine PNCT-spiked beverage samples without pretreatment, in addition to a kind of PAP-containing drug. The recoveries were validated using high performance liquid chromatography (HPLC). The results revealed that the developed GNP-ICS assay was reliable for the detection of PNCT in practical samples.

Yong Shi,1,&ast; Deru Meng,1,&ast; Shuanghu Wang,1 Peiwu Geng,1 Tao Xu,2 Quan Zhou,1 Yunfang Zhou,1 Wanshu Li,3 Xugao Chen4 1Comprehensive Breast Health Center, Department of Thyroid and Breast Surgery, The Sixth Affiliated Hospital of Wenzhou Medical University, The People’s Hospital of Lishui, Lishui, 323000, People’s Republic of China; 2Department of Pharmacy, Ningbo First Hospital, Ningbo, 315010, Zhejiang, People’s Republic of China; 3Department of Pharmacy, Ningbo Municipal Hospital of Traditional Chinese Medicine, Ningbo, 315010, Zhejiang, People’s Republic of China; 4Department of Radiology, The Sixth Affiliated Hospital of Wenzhou Medical University, The People’s Hospital of Lishui, Lishui, 323000, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Xugao ChenDepartment of Radiology, The Sixth Affiliated Hospital of Wenzhou Medical University, The People’s Hospital of Lishui, Lishui, 323000, People’s Republic of ChinaTel/Fax +86578-2780081Email lishui001@163.comWanshu LiDepartment of Pharmacy, Ningbo Municipal Hospital of Traditional Chinese Medicine, Ningbo, 315010, Zhejiang, People’s Republic of ChinaTel/Fax +86574-87089100Email wanshu_lws@126.comPurpose: Avitinib is the first third-generation epithelial growth factor receptor (EGFR) inhibitor independently developed in China and is mainly used for treating non-small cell lung cancer. However, pharmacokinetic details are limited. This study explored the in vivo and in vitro effects of avitinib on cytochrome CYP450 enzymes metabolic activity.Methods: A rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for determining six probe substrates and their metabolites. Avitinib influence on activity levels of CYP isozymes was examined in vitro using human and rat liver microsomes (HLMs/RLMs). For in vivo studies, rats were pretreated with 30 mg/kg avitinib once daily for 7 days (avitinib multiple-doses group), 30 mg/kg avitinib on day 7 (avitinib single-dose group), or an equivalent amount of CMC-Na once daily for 7 days (control group), followed by intragastrical administration of the probe substrates (1 mg/kg tolbutamide and 10 mg/kg phenacetin, bupropion, chlorzoxazone, dextromethorphan, and midazolam). Plasma pharmacokinetics and IC50 values of the probe substrates were then compared. Pharmacokinetic parameters were determined using non-compartmental analysis implemented in a pharmacokinetic program.Results: In vitro experiments revealed different inhibitory effects of avitinib on the six probe substrates with various IC50 values (bupropion, 6.39/22.64 μM; phenacetin, 15.79/48.36 μM; chlorzoxazone, 23.15/57.09 μM; midazolam, 27.64/59.6 μM; tolbutamide, 42.18/6.91 μM; dextromethorphan, 44.39/56.57 μM, in RLMs and HLMs respectively). In vivo analysis revealed significant differences (P < 0.05) in distinct pharmacokinetic parameters (AUC(0-t), AUC (0-∞), Cmax, MRT(0-t), MRT (0-∞), and CLz/F) for the six probe substrates after avitinib pretreatment.Conclusion: A sensitive and reliable UPLC-MS/MS method was established to determine the concentration of six probe substrates in rat plasma. Avitinib had inhibitory effects on CYP450 enzymes, especially cyp2b1, cyp1a2 in RLMs, CYP2C9 in HLMs, and cyp1a2, cyp2b1, cyp2d1, and cyp2e1 in vivo. Our data recommend caution when avitinib was taken simultaneously with drugs metabolized by CYP450 enzymes.Keywords: cytochrome, EGFR inhibitor, UPLC-MS/MS, rat liver microsome, drug-drug interaction

Recent studies indicated obvious impacts of nano-carriers on cytochrome P450 enzymes (CYP450s) in vitro , but the effects in vivo are still unknown. In the present research, mPEG 2k -PCL x micelles with different length of hydrophobic block (2000–10,000 Da) were intravenously administrated into rats for 14 days to evaluate the sub-chronic influences in the metabolic function of hepatic CYP450s. Although CYP1A1/B2 was susceptible to mPEG 2k -PCL x micelles compared with other CYP isoenzymes, induction was mainly observed and varied with micelle type, administration dose, and CYP isoform. Interestingly, mPEG 2k -PCL 3.5k micelles at 5 mg/kg increased the activity of CYP1A2, CYP2B1, CYP2C6, CYP2C11, and CYP3A1/2 while mPEG 2k -PCL 5k micelles only induced the latter three enzymes at 75 mg/kg. The mRNA expression of corresponding CYPs was mostly up-regulated by mPEG 2k -PCL 3.5k micelles whilst less effect in protein level except for CYP3A1/2. Moreover, mPEG 2k -PCL 3.5k micelles could affect the pharmacokinetic properties of phenacetin (CYP1A2), tolbutamide (CYP2C6), omeprazole (CYP2C11), and midazolam (CYP3A1/2) with a decrease of 19.6% in C max , 20.5% in AUC 0-t , 31.6% in AUC 0-t , and 40.1% in C max at 5 mg/kg, respectively ( P P

This study developed an open-tubular capillary electrochromatography protocol for the analysis of antipyretic analgesic drugs, which used a multifunctional homopolymer as coating. A controlled/living radical polymerization strategy was adopted to obtain poly(N-acryloxysuccinimide) with a tunable chain-length. The homopolymer coating enhanced the separation performance by contributing to the hydrophobic and hydrogen-bonding interactions between the analytes and the homopolymer. The effect of polymer chain-length and buffer pH and concentration on the separation efficiency was evaluated. In this approach, baseline separation of the three test drugs was achieved within 15 min. The repeatability of the prepared homopolymer coating was investigated, with the relative standard deviations < 2.88% observed in intra- and interday runs. Good linearity in the 5-800 µM range (R2 ≥ 0.998) demonstrates that accurate quantitative analysis of real samples was achieved. Moreover, the proposed assay was used to quantify the three drugs (aminopyrine, 4-aminoantipyrine, and phenacetin) in urine samples, achieving recovery rates between 92.1 and 108.7%. This promising methodology may be used for the analysis of drugs in real bio-samples and for the development of unique homopolymer coatings for open-tubular capillary electrochromatography systems.

Activated carbon obtained from ayous sawdust, Cucurbitaceae (egussi) peelings and the mixture of the two were studied for the adsorption of phenacetin. Characterisation of activated carbon by SEM and XRD analysis shows that the mixture of precursors combine the properties of activated carbon obtained separately. The well-knownbatch sorption models– Langmuir (one and two sites), Freudlich, Tempkin, Elovich, Langmuir-Freudlich, Redlich Peterson, Radke-Prausnitz, Fritz Shlunder)—were tested with experimental data for the adsorption of phenacetin to estimate adsorption equilibrium parameters—rate constantsand adsorption capacities. The model with the best fit was identified from extensive statistical analysis of the results of nonlinear regression of the experimental data. Comparison of the statistical errors in parameter estimation between linear and non-linear isotherm models shows that transformation of non-linear isotherm equations to linear forms implicitly alter their error structure. The much smaller size of the various error indicators —Determination Coefficient, R2; Sum of Square Errors, SSE; Chi Test, χ2; Average Relative Errors, ARE—, calculated for the case of non linearization when compared to linearization, indicate the greater accuracy in the application of non linearization. The Langmuir model (one site) gave the best fit and thus the values of adsorption capacity for each activated carbon were calculated from it. Kinetic models show that weak and strong interactions are involved in the adsorption process and that the controlling mechanism may not be limited to intra particle diffusion. The lower value of the boundary layer thickness in the case of activated carbon obtained from the mixture, justified the higher adsorbed quantity of this activated carbon compared to those of activated carbon from each precursor.

This study aimed to determine simultaneously five major street cocaine adulterants (caffeine, lidocaine, phenacetin, diltiazem, and hydroxyzine) in human urine by dispersive liquid-liquid microextraction (DLLME) and high-performance liquid chromatography. The chromatographic separation was obtained in gradient elution mode using methanol:water plus trifluoroacetic acid 0.15% (v/v) (pH = 1.9) at 1 mL min−1 as mobile phase, at 25 °C, detection at 235 nm, and analysis time of 20 min. The effect of major DLLME operating parameters on extraction efficiency was explored using the multifactorial experimental design approach. The optimum extraction condition was set as 4 mL human urine sample alkalized with 0.5 M sodium phosphate buffer (pH 12), NaCl (15%, m/v), 300 μL acetonitrile (dispersive solvent), and 800 μL chloroform (extraction solvent). Linear response (r2 ≥ 0.99) was obtained in the range of 180–1500 ng mL−1 with suitable selectivity, quantification limit (180 ng mL−1), mean recoveries (33.43–76.63%), and showing relative standard deviation and error (within and between-day assays) ≤15%. The analytes were stable after a freeze-thaw cycle and a short-term room temperature stability test. This method was successfully applied in real samples of cocaine users, suggesting that our study may contribute to the appropriate treatment of cocaine dependence or with the cases of cocaine acute intoxication.

Cocaine is usually sold as a white powder and can contain several adulterants and diluents, known as cutting agents. The cutting agents play an important role in the identification of trafficking routes, and they can also modify or intensify signs and symptoms of drug intoxication increasing the risk to the health's user. The purpose of this work was to quantify cocaine and cutting agents in 116 illicit samples from NMS Labs, Willow Grove, PA, U.S. Gas chromatography - mass spectrometry (GC-MS) and handle-portable gas chromatography toroidal ion trap mass spectrometry (GC-TMS) were used as screening methods A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of cocaine, levamisole, benzocaine, phenacetin, hydroxyzine, theophylline, diltiazem, acetaminophen and caffeine. Cocaine-d3 and caffeine-d3 were used as internal standards. The method was shown to be precise, accurate and linear over a range of 50-2000ng/mL for all analytes. Cocaine was the only detected compound in 16.37% (n=19) of the samples. Between the identified cutting agents, levamisole was the most abundant substance found (79.31% of the total samples, amounts ranging from 0.2 to 74.3%), followed by phenacetin (18.96%, 0.3-46.8%), caffeine (12.06%, 0.2-32.2%), hydroxyzine (9.48%, 0.7-13.8%) and benzocaine (5.17%, 0.4-58.3%). GC-TMS was considered suitable to be used as a tool in forensic analysis as a screening method for cocaine, benzocaine, phenacetin, hydroxyzine and caffeine with restrictions to be used for levamisole, while GC-MS presented good results in screening analysis for cocaine, levamisole, benzocaine, phenacetin, hydroxyzine and caffeine.

A high incidence of positive results is obtained with in vitro genotoxicity tests, which do not correlate with the in vivo negative results in many cases. To address this issue, the metabolic profile of rat liver 9000 × g supernatant fraction (S9) pretreated with phenobarbital (PB) and 5,6-benzoflavone (BNF) was characterized. Furthermore, the in vitro micronucleus tests of 10 compounds were performed with PB-BNF-induced rat S9. PB-BNF increased cytochrome P450 (CYP) activity and CYP1A1, CYP1A2, CYP2B1/2, CYP2C6, CYP3A1, and CYP3A2 expression in rat S9, whereas it decreased CYP2C11 and CYP2E1 expression. PB-BNF-induced S9 enhanced the micronucleus induction (MI) of benzo[a]pyrene (BaP), cyclophosphamide (CPA), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine hydrochloride (PhIP), which are metabolized by CYP1A1, CYP2C6, and CYP1A2, respectively. In contrast, coumarin and chlorpheniramine showed MI with PB-BNF-induced S9 despite the fact that they show negative results in the in vivo studies. Furthermore, diclofenac, piroxicam, lansoprazole, and caffeine showed MI regardless of the enzyme induction by PB-BNF, whereas phenacetin did not show MI. These results indicate that PB-BNF-induced rat S9 is effective in detecting the genotoxic potential of promutagens, such as BaP, CPA, and PhIP, but not of coumarin and chlorpheniramine, probably due to the differences in the in vitro and in vivo metabolic profile and its exposure levels of the drugs.

Nyaope, a street drug commonly found in South Africa, is a mixture of low grade heroin, cannabis products, antiretroviral drugs and other materials added as cutting agents. It is a highly physiologically addictive substance which is smoked by users. Little work has been published on the chemical analysis and profiling of nyaope. Sample preparation prior to chromatographic or spectrometric analysis normally involves dissolution of the sample in an organic solvent. This study determined the most suitable organic solvent in which the common components of nyaope, namely Δ9-tetrahydrocannabinol, diamorphine, caffeine, dextromethorphan, phenacetin and the antiretrovirals efavirenz and nevirapine, which have different chemical characteristics, are stable during extraction and prior to analysis of nyaope samples i.e. autosampler stability. Street samples of cannabis (Δ9-tetrahydrocannabinol), heroin (diamorphine) and antiretrovirals were mixed to mimic a nyaope sample and dissolved in the organic solvents dichloromethane, ethanol, ethyl acetate, hexane, isopropanol and tertiary butyl alcohol. Analysis was performed after intervals of 0, 1, 6, 8, 24, 48 and 72 h, prior to analysis by gas chromatography–mass spectrometry. Tertiary butyl alcohol resulted in the most stable extracts of the main nyaope components after 72 h of storage. The analysis was also repeated on actual street samples of nyaope. These results show that tertiary butyl alcohol is a suitable solvent for sample preparation for the identification, comparison and profiling of nyaope samples.

Przybyłek, M.; Kowalska, A. Tymorek, N.; Dziaman, T.; Cysewski, P. Thermodynamic Characteristics of Phenacetin in Solid State and Saturated Solutions in Several Neat and Binary Solvents. Molecules 2021, 26, 4078. https://doi.org/10.3390/ molecules26134078 ABSTRACT:The thermodynamic properties of phenacetin in solid state and in saturated conditions in neat and binary solvents were characterized based on differential scanning calorimetry and spectroscopic solubility measurements. The temperature-related heat capacity values measured for both the solid and melt states were provided and used for precise determination of the values for ideal solubility, fusion thermodynamic functions, and activity coefficients in the studied solutions. Factors affecting the accuracy of these values were discussed in terms of various models of specific heat capacity difference for phenacetin in crystal and super-cooled liquid states. It was concluded that different properties have varying sensitivity in relation to the accuracy of heat capacity values. The values of temperature-related excess solubility in aqueous binary mixtures were interpreted using the Jouyban–Acree solubility equation for aqueous binary mixtures of methanol, DMSO, DMF, 1,4-dioxane, and acetonitrile. All binary solvent systems studied exhibited strong positive non-ideal deviations from an algebraic rule of mixing. Additionally, an interesting co-solvency phenomenon was observed with phenacetin solubility in aqueous mixtures with acetonitrile or 1,4-dioxane. The remaining three solvents acted as strong co-solvents.

Poziotinib is an orally active, irreversible, pan-HER tyrosine kinase inhibitor used to treat non-small cell lung cancer, breast cancer, and gastric cancer. Poziotinib is currently under clinical investigation, and understanding its drug-drug interactions is extremely important for its future development and clinical application. The cocktail method is most suitable for evaluating the activity of cytochrome P450 enzymes (CYPs). As poziotinib is partially metabolized by CYPs, cocktail probes are used to study the interaction between drugs metabolized by each CYP subtype. Midazolam, bupropion, dextromethorphan, tolbutamide, chlorzoxazone, phenacetin, and their metabolites were used to examine the effects of poziotinib on the activity of cyp1a2, 2b1, 2d1, 2c11, 2e1, and 3a1/2, respectively. The in vitro experiment was carried out by using rat liver microsomes (RLMs), whereas the in vivo experiment involved the comparison of the pharmacokinetic parameters of the probes after co-administration with poziotinib to rats to those of control rats treated with only probes. UPLC-MS/MS was used to detect the probes and their metabolites in rat plasma and rat liver microsomes. The in vitro results revealed that the half-maximal inhibitory concentration values of bupropion and tolbutamide in RLMs were 8.79 and 20.17 μM, respectively, indicating that poziotinib showed varying degrees of inhibition toward cyp2b1 and cyp2c11. Poziotinib was a competitive inhibitor of cyp2b1 and cyp2c11, with Ki values of 16.18 and 17.66 μM, respectively. No time- or concentration-dependence of inhibition by poziotinib was observed toward cyp2b1 and cyp2c11 in RLMs. Additionally, no obvious inhibitory effects were observed on the activity of cyp1a2, cyp2d1, cyp2e1, and cyp3a1/2. In vivo analysis revealed that bupropion, tolbutamide, phenacetin, and chlorzoxazone showed significantly different pharmacokinetic parameters after administration (p < 0.05); there was no significant difference in the pharmacokinetic parameters of dextromethorphan and midazolam. These results show that poziotinib inhibited cyp2b1 and cyp2c11, but induced cyp1a2 and cyp2e1 in rats. Thus, poziotinib inhibited cyp2b1 and cyp2c11 activity in rats, suggesting the possibility of interactions between poziotinib and these CYP substrates and the need for caution when combining them in clinical settings.

Phenacetin (PNCT), a common antipyretic and analgesic drug, is often used to treat fever and headache. However, the effect of PNCT on nitrifiers in wastewater treatment processes remains unclear. The practicability of attaining partial nitrification (PN) through inhibitor-PNCT was investigated in this study. The optimal treatment conditions of soaking once for 18 h with 2.50 × 10−3 g PNCT/(g MLSS) were applied to the PN stability experiment. The results showed that ammonia oxidation activity recovered quickly after 3 cycles of operation, while nitrite oxidation activity was suppressed steadily. In addition, average ammonium removal efficiency and nitrite accumulation ratio during 138 cycles could reach 94.94% and 85.38%, respectively. Complimentary DNA high-throughput sequencing and oligotyping analysis showed that the activity of Nitrosomonas would gradually surpass Nitrospira after PNCT treatment only once. The decrease of Nitrospira activity was accompanied by the simplification of oligotypes after PNCT treatment, while Nitrosomonas could adapt to PNCT stress by reducing the differences between oligotypes. Metagenomics revealed that the decrease in the number of NXR in the nitrogen metabolism pathways was the key reason for achieving PN. The potential mechanisms might be that the dominant nitrite-oxidizing bacteria and complete ammonia oxidizers were bio-killed by PNCT.

High-performance liquid chromatography (HPLC) is the most common analytical method practiced in various fields and used for analysis of almost all drug compounds in the pharmaceutical industries. During drug development, an evaluation of potential drug interaction with cytochrome P450 (CYP) is essential. A “cocktail” approach is often used in drug development to evaluate the effect of a drug candidate on multiple CYP enzymes in a single experiment. So far, simultaneous analysis of multiple CYP substrates, which have greatly different structure and physicochemical properties, has required organic solvents and mobile phase gradient methods. However, despite the recent emphasis on environmental protection, analytical methods that use only aqueous solvents without the use of organic solvents for separation have not been studied well. This study sought to develop the simultaneous analysis of multiple CYP substrates by using poly(N-isopropylacrylamide) (PNIPAAm)-based temperature-responsive chromatography with only aqueous solvents and isocratic methods. Good separation of multiple CYP substrates was achieved without using organic solvents and any gradient methods by temperature-responsive chromatography utilizing a P(NIPAAm-co-n-butyl methacrylate (BMA))- and P(NIPAAm-co-N-acryloyl L-tryptophan methyl ester (L-Trp-OMe))-grafted silica column. Overall, PNIPAAm-based temperature-responsive chromatography represents a remarkably simple, versatile, and environmentally friendly bioanalytical method for CYP substrates and their metabolites.

Introduction Ex vivo studies of human fetal hepatic drug metabolism are uncommon as it requires access to functional liver tissue and therefore raises practical and ethical concerns. Large animal models provide an alternative opportunity to study changes in cytochrome P450 (CYP) activity in the mother and fetus during pregnancy. We aimed to develop methods to determine the activity of CYP1A2, CYP2C9, CYP2D6 and CYP3A4 in sheep hepatic microsomes. Methods We identified optimal conditions to determine the activity of CYP1A2 (using the probe drug phenacetin), CYP2C9 (diclofenac), CYP2D6 (dextromethorphan) and CYP3A4 (midazolam) by varying techniques for microsome extraction, probe drug concentration, incubation time and microsome concentration. The specificity of each probe drug was assessed by determining the rate of metabolism when specific CYP enzyme inhibitors were included in the reaction. Results The optimum incubation time and probe drug concentration was six hours with 5 μM phenacetin (CYP1A2), four hours with 10 μM diclofenac (CYP2C9), 30 min with 1 μM of midazolam (CYP3A4) and 10 min with 1 μM dextromethorphan (CYP2D6). For both CYP2D6 and CYP3A4 reactions required 20 μg of microsomal protein, whereas for CYP1A2 and CYP2C9, reactions required 40 μg of microsomal protein. Metabolism of phenacetin, dextromethorphan and midazolam was reduced by specific enzyme inhibitors, but the specific CYP2C9 inhibitor sulfaphenazole did not substantially inhibit diclofenac metabolism. Discussion This study identifies the optimal conditions for determining CYP activity in maternal sheep hepatic microsomes. In doing so, we have developed a standardised protocol for assessment of microsomal activity of CYP3A4, CYP1A2 and CYP2D6, but we were unable to optimise conditions for assessment of CYP2C9. This approach can be applied to investigate the impact of pregnancy complications on maternal and fetal hepatic drug metabolism.

Background: Vonoprazan fumarate is a potassium-competitive acid blocker that was developed as a novel acid-suppressing drug for multiple indications. As a potential alternative to proton-pump inhibitors, the determination of the drug-drug interactions is vital for further applications. Probe drug cocktails are a type of rapid, economical and efficient approach for evaluating cytochrome P450 enzyme activities. Since vonoprazan is metabolized partly by cytochrome P450, cocktails were used to study CYP-based drug-drug interactions. Methods: This study was conducted both in vitro and in vivo. In the in vitro study of rat liver microsomes, ultra-performance liquid chromatography coupled to tandem mass spectrometry was utilized to assess the reversible inhibition of cytochrome P450 by vonoprazan by determining the concentration of probe drugs (phenacetin, bupropion, tolbutamide, dextromethorphan, midazolam, chlorzaoxazone). The differences in the levels of probe drugs between the rat groups with or without vonoprazan administration were also tested in the rats. Results: In vitro analysis revealed that the IC50 values of midazolam, tolbutamide, dextromethorphan, and bupropion in rat microsomes were 22.48 μM, 18.34μM, 3.62μM, and 3.68μM, respectively, while chlorzoxazone and phenacetin displayed no inhibition. In vivo analysis revealed that midazolam, bupropion, dextromethorphan, and tolbutamide showed significant (P

The cutting agents, classified as diluents (pharmacologically inactive) or adulterants (pharmacologically active), are substances commonly used to cut drugs of abuse to increase profits. These substances are constantly changing over time, increasing the risks to the user's health caused by the compounds' potential individual toxicities as well as their drug-drug interactions. This work aimed to develop and validate a screening method using a portable quadrupole-based gas chromatography mass spectrometer (FLIR Griffin™ G510) to identify drugs of abuse and adulterants in seized material, and compare it with a well validated standard technology, gas chromatography mass spectrometry (GC-MS). The method was validated for the identification of alprazolam, amphetamine, aminopyrine, benzocaine, caffeine, cocaine, codeine, diltiazem, ephedrine, fentanyl, fenethylline, furanylfentanyl, heroin, hydroxyzine, levamisole, lidocaine, methamphetamine, morphine, noramidopyrine (a marker of metamizole), phencyclidine, phenacetin, procaine, strychnine and xylazine. The targeted substances were chosen based on current intelligence regarding prevalent adulterants observed in multiple jurisdictions. Interference, precision, robustness and carryover were evaluated. The method was successfully validated and proved to be suitable to detect and identify the 24 target compounds proposed. The reliability of the instrument for detecting the presence of targeted compounds was analyzed by using Receiver Operating Characteristic (ROC) analysis. The portable quadrupole-based gas chromatography mass spectrometer was considered suitable for use in forensic analysis as a screening method.

Red ginseng is often combined with Aconiti Lateralis Radix Praeparata to reduce alkaloids-related toxicity of the latter. Such herb-pairing also results in better therapeutic effect in heart failure, as compared to the singular use of either herb. The purpose of this study was to investigate the effect of Aconiti Lateralis Radix Praeparata and its combination with red ginseng on the activities of CYP450 enzymes in rats. A sensitive and reliable HPLC-MS/MS method was established and validated for the simultaneous determination of eight probe drugs, phenacetin (CYP1A2), tolbutamide (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), dapsone (CYP3A4), 7-hydroxycoumarin (CYP2A6), bupropion (CYP2B6), and amodiaquine (CYP2C8), in rat plasma using diazepam as internal standard (IS). The chromatographic separation was performed on a Waters XBridge™ C18 column (2.1 mm × 100 mm, 3.5 μm) using a gradient elution with the mobile phase consisting of acetonitrile and water (containing 0.1% formic acid) at a flow rate of 0.3 mL/min. The method was successfully applied in evaluating the effect of Aconiti Lateralis Radix Praeparata and red ginseng on the activities of CYP450 enzymes. The pharmacokinetic results of the eight probe drugs suggested that Aconiti Lateralis Radix Praeparata may inhibit the activity of CYP2A6, CYP2C19, CYP2B6, CYP1A2, CYP3A4, and CYP2C9 enzymes in rats. Comparison between the two groups, Aconiti Lateralis Radix Praeparata combined with red ginseng and Aconiti Lateralis Radix Praeparata, indicated that red ginseng may inhibit the activity of CYP2D6 and CYP2B6 enzymes while inducing the activity of CYP1A2, CYP3A4, and CYP2C9 enzymes.

A new method, simple and fast, for fluconazole (FLU) quantification in cerebrospinal fluid (CSF) samples using dispersive liquid-liquid microextraction (DLLME) and an eco-friendly mobile phase for HPLC-PDA was developed. The study of DLLME extraction condition covered the investigation of 12 combinations of extraction and disperser solvents followed by a fractional factorial design 2(7-3) to determine the influence of seven factors. After this stage, a central composite design was performed for three factors and a response surface was obtained. Aiming a compromise between a good recovery and a low organic solvent use it was established an extraction condition that consists of: 100 μL of chloroform, 100 μL of isopropyl alcohol, 200 μL of CSF, 200 μL of 50 mM phosphate buffer pH 7.3 and centrifugation for 5 min at 2200g and 4 °C. The HPLC analysis used an Ascentis® Express C18 column (100 mm × 4.6 mm, 2.7 μm) and an Ascentis® Express C18 guard column (3 mm × 4.6 mm, 2.7 μm), ethanol : water (15 : 85, v/v) as mobile phase, temperature of 45 °C, flow rate of 0.8 mL min-1 and phenacetin as internal standard. The method validation was performed according to European Agency's Guideline on Bioanalytical Validation Methodology and a linear range was obtained from 0.25 to 62.5 μg mL-1, with precision and accuracy within the recommended limits and recovery of 70% for FLU and 81% for phenacetin. Samples were stable in the studies performed and the method showed to be selective and with no carryover effect. The feasibility of the obtained method was confirmed by FLU determination at a CSF from a patient who was treated for neuromycosis. Therefore, here is described a method that meets many principles of green analytical chemistry and is useful for FLU therapeutic monitoring.

A role of the gut microbiota in influencing brain function and emotional disorders has been suggested. However, only a few studies have investigated the gut microbiota in the context of drug addiction. Cocaine can be smoked (i.e., crack or coca paste) and its consumption is associated with a very high abuse liability and toxicity. We have recently reported that cocaine base seized samples contained caffeine and phenacetin as main active adulterants, which may potentiate its motivational, reinforcing, and toxic effects. However, the effect of volatilized cocaine and adulterants on the gut microbiota remained unknown. In the present study, we evaluated the effect of volatilized cocaine and two adulterants on the structure, diversity, and functionality of the gut microbiota in rats. Animals were chronically exposed to the fume of cocaine, caffeine, and phenacetin during 14 days. At the end of the treatment, feces were collected and the structure, composition, and functional predictions of the gut microbiota were analyzed. Cocaine significantly decreased the community richness and diversity of the gut microbiota while both cocaine and phenacetin drastically changed its composition. Phenacetin significantly increased the Firmicutes-Bacteroidetes ratio compared to the control group. When the predicted metagenome functional content of the bacterial communities was analyzed, all the treatments induced a dramatic decrease of the aromatic amino acid decarboxylase gene. Our findings suggest that repeated exposure to volatilized cocaine, as well as to the adulterants caffeine and phenacetin, leads to changes in the gut microbiota. Future studies are needed to understand the mechanisms underlying these changes and how this information may support the development of novel treatments in drug addiction.

The aim of the present study was to investigate the potential effect of thymoquinone (TQ) on the metabolic activity of four major drug metabolizing enzymes in human liver microsomes, namely cytochrome P450 (CYP) 1A2, CYP2C9, CYP2D6 and CYP3A4. The inhibition of CYP enzymatic activities by TQ was evaluated by incubating typical substrates (phenacetin for CYP1A2, tolbutamide for CYP2C9, dextromethorphan for CYP2D6, and testosterone for CYP3A4) with human liver microsomes and NADPH in the absence or presence of TQ (1, 10 and 100 µM). The respective metabolite of the substrate that was formed was measured by HPLC. Results of the presented study presented that the metabolic activities of all the investigated CYP enzymes, viz. CYP1A2, CYP2C9, CYP2D6 and CYP3A4, were inhibited by TQ. At 1 µM TQ, CYP2C9 enzyme activity was maximally inhibited by 46.35%, followed by CYP2D6 (20.26%) > CYP1A2 (13.52%) > CYP3A4 (12.82%). However, at 10 µM TQ, CYP2C9 enzyme activity was maximally inhibited by 69.69%, followed by CYP3A4 (23.59%) > CYP1A2 (23.51%) > CYP2D6 (11.42%). At 100 µM TQ, CYP1A2 enzyme activity was maximally inhibited by 81.92%, followed by CYP3A4 (79.24%) > CYP2C9 (69.22%) > CYP2D6 (28.18%). The IC50 (mean ± SE) values for CYP1A2, CYP2C9, CYP2D6 and CYP3A4 inhibition were 26.5 ± 2.9 µM, 0.5 ± 0.4 µM, >500 µM and 25.2 ± 3.1 µM, respectively. These findings suggest that there is a high probability of drug interactions resulting from the co-administration of TQ or herbs containing TQ with drugs that are metabolized by the CYP enzymes, particularly CYP2C9. Keywords: Thymoquinone, Cytochrome P450, Metabolism, Human liver microsomes

To improve the efficiency of drug-discovery research on pyrrole-imidazole polyamides (PIs), a more rapid method for quantitative and qualitative measurement of PI in rat plasma samples was developed here using ultra-fast liquid chromatography-ultraviolet spectrometry (UFLC-UV) in order to shorten the measurement time. A measurement method of PIs by HPLC developed until now takes 45 min for one sample measurement. This method was inefficient to investigate extraction conditions from biological samples and measurement of animal experimental samples. In the developed method of this study, PI and phenacetin (internal standard, IS) were separated with an ACQUITY UPLC HSS T3 (1.8 µm, 2.1 × 50 mm; Nihon Waters K.K., Japan) column using a mobile phase of 0.1% acetic acid (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 0.3 mL/min with a linear gradient. The detection wavelength was 310 nm. The calibration curve was linear in the range of 0.225-4.5 µg/mL (correlation coefficients ≥0.9995, n = 5). The intra- and inter-day accuracies were in the range of -6.04 to 12.2%, and the precision was less than 2.99%. The measurement time of this method (7 min per injection) was markedly shortened to about one-sixth of the previous measurement time (45 min per injection). This is the first report describing the quantitative and qualitative measurement of PI in plasma using UFLC-UV. The present method will be very useful for the drug-discovery research of PIs.

The inhibitory effect of new chemical entities on rat liver P450 marker activities was investigated in a functional approach towards drug development. Treatment of colorectal cancer (CRC) and chemoprevention using salicylic acid has gained a lot of attention, mainly in the prevention of the onset of colon cancer. Thus, an in vitro inhibitory effect of salicylic acid on rat CYP2C11 activity was examined by using high performance liquid chromatography (HPLC). High performance liquid chromatography analysis of a CYP2C11 assay was developed on a reversed phase C18 column (SUPELCO 25 cm &times, 4.6 mm &times, 5 &micro, m) at 243 nm using 32% phosphate buffer (pH 3.36) and 68% methanol as a mobile phase. The CYP2C11 assay showed good linearity for all components (R2 &gt, 0.999). Substrates and metabolites were found to be stable for up to 72 hours. Additionally, the method demonstrated good reproducibility, intra- and inter-day precision (&lt, 15%), acceptable recovery and accuracy (80%&ndash, 120%), and low detection (1.3501 &micro, M and 3.2757 &micro, M) and quantitation limit values (4.914 &micro, M and 9.927 &micro, M) for 16&alpha, hydroxytestosterone and testosterone, respectively. Salicylic acid acts reversibly as a noncompetitive (weak) inhibitor with Ki = 84.582 &plusmn, 2.67 &micro, M (concentration of inhibitor to cause 50% inhibition of original enzyme activity (IC50) = 82.70 &plusmn, M) for CYP2C11 enzyme activity. This indicates a low potential to cause toxicity and drug&ndash, drug interactions.

Cytochrome P450 (CYP) 1A2, 2C9, 2D6, and 3A4 are the most important phase I drug-metabolizing enzymes in the liver, but there is a dearth of literature available on the effects of naltrexone hydrochloride on these major enzymes present in the human liver. Thus, in the present study, the effect of naltrexone hydrochloride on the activity of CYP1A2, 2C9, 2D6, and 3A4 using human liver microsomes (HLM) was investigated. A selective probe for CYP1A2, 2C9, 2D6, and 3A4 was incubated with HLM with or without naltrexone hydrochloride. Phenacetin O-deethylation, tolbutamide 4-hydroxylation, dextromethorphan O-demethylation, and testosterone 6β-hydroxylation reactions were monitored for enzyme activity. The activity of all the studied CYP enzymes except 1A2 was significantly inhibited by naltrexone hydrochloride 1 µM. Furthermore, 1 µM naltrexone hydrochloride inhibited CYP3A4 enzyme activity, the most by 37.9% followed by CYP2C9 (36.5%) and CYP2D6 (31.8%). The CYP2C9 and CYP2D6 metabolic activities were greatly affected by naltrexone hydrochloride, which even at the lowest concentration of naltrexone hydrochloride (0.01 µM) significantly decreased the metabolic activity by 34.9 and 16.0%, respectively. The half maximal inhibition concentration (IC50) values for CYP2C9 and CYP2D6 inhibition were 3.40 ± 1.78 and 5.92 ± 1.58 µM, respectively. These outcomes advocate that there is a great possibility of drug interactions resulting from the concurrent administration of naltrexone hydrochloride with actives that are metabolized by these CYP enzymes, particularly CYP2C9 and CYP2D6. Nevertheless, further clarification is needed through detailed in vivo pharmacokinetic studies.

Single-walled carbon nanotubes (SWCNTs) are made from a rolled single sheet of graphene with a diameter in the nanometer range. SWCNTs are potential carriers for drug delivery systems because antibodies or drugs can be loaded on their surface; however, their effect on the activities of cytochrome P450 (CYP) remains unclear. The aim of this study was to investigate the effect of two kinds of SWCNTs with different lengths (FH-P- and SO-SWCNTs) on human CYP activity. In addition, other nano-sized carbon materials, such as carbon black, fullerene-C60 , and fullerene-C70 were also evaluated to compare their effects on CYP activities. Ten CYP substrates (phenacetin, coumarin, bupropion, paclitaxel, tolbutamide, S-mephenytoin, dextromethorphan, chlorzoxazone, midazolam, and testosterone) were used. Testosterone 6β-hydroxylation and midazolam 1'-hydroxylation, which are catalysed by both CYP3A4 and CYP3A5 in liver microsomes, were decreased by 25% and 45%, respectively, in the presence of 0.1 mg/ml SO-SWCNT. Dextromethorphan O-demethylation, which is catalysed mainly by CYP2D6, was decreased by 40% in the presence of SO-SWCNT. Other CYP activities, however, were not attenuated by SO-SWCNT. FH-P-SWCNT, carbon black, fullerene-C60 , and fullerene-C70 at 0.1 mg/ml had no effect on CYP activities. The Ki values for testosterone 6β-hydroxylation, midazolam 1'-hydroxylation, and dextromethorphan O-demethylation in liver microsomes were 136, 34, and 56 μg/ml, respectively. SO-SWCNT was determined to be a competitive inhibitor of CYP3A4, CYP3A5, and CYP2D6. These results suggest that the effect of SO-SWCNT differs among CYP isoforms, and that the inhibition potency depends on the physicochemical properties of the nanocarbons.

Adulteration is a common practice in the illicit drugs market, but the psychoactive and toxic effects provided by adulterants are clinically underestimated. Coca-paste (CP) is a smokable form of cocaine which has an extremely high abuse liability. CP seized samples are sold adulterated; however, qualitative and quantitative data of CP adulteration in forensic literature is still scarce. Besides, it is unknown if adulterants remain stable when CP is heated. This study was designed to report the chemical content of an extensive series of CP seized samples and to demonstrate the stability (i.e., chemical integrity) of the adulterants heated. To achieve this goal, the following strategies were applied: (1) a CP adulterated sample was heated and its fume was chemically analyzed; (2) the vapor of isolated adulterants were analyzed after heating; (3) plasma levels of animals exposed to CP and adulterants were measured. Ninety percent of CP seized samples were adulterated. Adulteration was dominated by phenacetin and caffeine and much less by other compounds (i.e., aminopyrine, levamisole, benzocaine). In the majority of CP analyzed samples, both cocaine and caffeine content was 30%, phenacetin 20% and the combination of these three components reached 90%. Typical cocaine pyrolysis compounds (i.e., BA, CMCHTs, and AEME) were observed in the volatilized cocaine and CP sample but no pyrolysis compounds were found after isolated adulterants heating. Cocaine, phenacetin, and caffeine were detected in plasma. We provide current forensic data about CP seized samples and demonstrated the chemical integrity of their adulterants heated.

The worldwide consumption of illicit drugs presents a big problem in terms of health care and prosecution. In the recent years, hundreds of novel psychoactive substances came up and were traded via the Internet, but there is still a big demand for classic illicit drugs such as cocaine, heroin, cannabis and ecstasy. Among these, cocaine particularly is frequently altered not only with excipients, but also with other physiologically active substances. The purpose of this work was to estimate the trend of cocaine purity and abundance of its adulterants in samples seized by Austrian police from 2012 to 2017. A micro-HPLC method for quantification of cocaine and its most common adulterants was developed and validated using gradient elution and UV detection at four wavelengths. 110 cocaine samples were analysed. In all the samples cocaine was present as hydrochloric salt. Caffeine, procaine, levamisole, phenacetin, lidocaine and benzocaine were the most abundant adulterants.

In this study, a new method was developed to quantify cocaine and some adulterants (lidocaine, caffeine, phenacetin, procaine and benzocaine) using nuclear magnetic resonance spectroscopy without the use of deuterated solvents (No-D qNMR). No-D qNMR presents as main advantages: low cost (in comparison to the use of deuterated solvents), non-destructive and rapid analysis, and being able to detect non-volatile compounds. For analytical validation achievement, figures of merit such as selectivity and specificity, linearity, quantification limit, detection limit, accuracy, precision and robustness were obtained. The built models presented excellent precision ( 0.99), and LOD values which varied between 0.126 and 0.666 mg mL−1. With the purpose of verifying the predictive capacity of the models, 34 crack samples seized by the Civil Police of Espirito Santo State – Brazil were analyzed, and the average cocaine content found was around 17.5 wt%, which is in line with expectations (up to approximately 30 wt%). Moreover, 50 wt% of the samples contained phenacetin, 9 wt% caffeine, and 3 wt% procaine and lidocaine, while benzocaine wasn't identified.

Aims P. quassioides is a traditional Chinese medicine used for the treatment of gastroenteritis, snakebite, infection and hypertension in China. 4, 5-dimethoxycanthin-6-one is one of the main active canthinone alkaloid isolated from P. quassioides . The aim of this work was to identify the cytochrome P (CYP) 450 enzymes responsible for the metabolism of 4, 5-dimethoxycanthin-6-one (DCO) and to evaluate the inhibitory effect of DCO on CYP activity in human liver microsomes (HLM) in vitro . Materials and methods the CYP isoforms responsible for DCO metabolism and the inhibitory effects of DCO on CYP activity was studied in HLM. Key findings The in vitro metabolic enzyme of DCO was CYP3A4 (mediated the formation of metabolites M1-M5), CYP2C9 (mediated the formation of metabolites M1-M3, M6 and M8) and CYP2D6 (mediated the formation of metabolite M3) in HLM. Furthermore, the present work found that DCO uncompetitively inhibited CYP1A2-mediated phenacetin O-deethylation with an IC 50 value of 1.7 μM and a Ki value of 2.6 μM. Significance The results suggested that the metabolic interaction should be existed when the substrate drugs of CYP1A2 were co-administered with DCO or traditional Chinese medicine containing it, such as the extract of P. quassioides and Kumu injection.

Gymnema sylvestre (GS) is a medicinal herb used for diabetes mellitus (DM). Herbs are gaining popularity as medicines in DM for its safety purpose. The aim of the present study was to evaluate in vivo pharmacokinetic (PK) interaction between allopathic drugs tolbutamide (TOLBU), amlodipine (AMLO), and phenacetin (PHENA) at low (L) and high (H) doses with ethanolic extract (EL) from GS. EL was extracted and subjected to TLC, total triterpenoid content (19.76 ± 0.02 W/W) and sterol content (0.1837 ± 0.0046 W/W) estimation followed by identification of phytoconstituents using HRLC-MS and GC-MS. PK interaction study with CYP2C9, CYP3A4 and CYP1A2 enzymes were assessed using TOLBU, AMLO and PHENA respectively to index cytochrome (CYP) mediated interaction in rats after concomitant administration of EL extract (400 mg/kg) from GS for 7 days. The rats were divided into four groups for each PK study where, group I and II were positive control for low and high dose of test drugs (CYP substrates) while group II and IV were orally administered EL. The PK study result of PHENA indicated that area under the plasma concentration–time curve (AUC0-24) was significantly (P

Aims Berberine (BER) is an important anti-bacterial drug from Chinese herbal medicine and a novel drug candidate for preclinical development in recent years. Here we provide evidence that the effects of berberine on cytochrome P450 (CYP) 1A2 in vitro and in vivo. Main methods Real-time polymerase chain reaction and western blotting analysis were employed to evaluate the CYP1A2 mRNA levels and protein expression. The enzyme activity was assessed by the metabolic rate of phenacetin to acetaminophen by LC-MS/MS method. Key findings The results indicated that the CYP1A2 mRNA expression and enzyme activity in HepG2 cells after treated with BER (4.5 μg/ml) exhibited a significant induction (16.11-fold and 5.0-fold, respectively), which was consistent with those on rat liver microsomes (4.5-fold and 1.98-fold, respectively) by BER induction (10 mg/kg/day, i.p.) ex vivo. Beside, BER induced CYP1A2 activity with increases in AUC0 − t and Cmax of acetaminophen and the Ke and t1/2 of phenacetin after oral administration of phenacetin (p Significance This study firstly reported the induction effect of BER on rats CYP1A2 by intraperitoneal route. But, BER didn't show significant induction effect on CYP1A2 by high-dose orally administrating to rats for 6 consecutive days due to the extremely low bioavailability. The potential drug-drug interactions were supposed to happen when the liver exposed to high dose of BER in vivo by changing administration route.

Acetaminophen (ACT) and phenacetin (PNT) removal during light-emitting diode (LED)-UV photolysis of persulfate (PS) was evaluated with a typical wavelength of 365 nm. Decay of PNT and ACT in pH ranges of 5.5–8.5 followed pseudo-first order kinetics. Maximum pseudo-first order rate constants (kobs) of ACT and PNT decomposition of 1.8 × 10−1 and 1.2 × 10−1 min−1, respectively, were obtained at pH 8.5. Hydroxyl radicals (·OH), sulfate radicals (SO4·-), superoxide radicals (O2−·), and singlet oxygen (1O2) were determined in-situ electron paramagnetic resonance (EPR) and alcohol scavenging tests. The average contributions of ·OH and SO4·- were 23.5% and 53.0% for PNT removal, and 15.9% and 53.0% for ACT removal at pH ranges of 5.5–8.5. In samples subjected to chlorination after LED-UV365/PS pre-oxidation, a relatively small total concentration of five halogenated disinfection by-products (DBPs) was obtained of 90.9 μg L−1 (pH 5.5) and 126.7 μg L−1 (pH 7.0), which is 58.5% and 30.2% lower than that in system without LED-UV365/PS pre-oxidation. Meanwhile, a higher maximum value of total DBP concentration was obtained at pH 8.5 (445.6 μg L−1) following LED-UV365/PS pre-oxidation. The results of economy evaluation showed that UV365 was more cost-effective in application for organic contaminant removal compared with UV254.

Porous graphene oxide (GO) adsorbents were successfully prepared by connecting GO sheets with tetrafluoroterephthalonitrile (TFT) or decafluorobiphenyl (DFB) through a nucleophilic aromatic substitution reaction. Textural characterization indicated that the enlarged surface area and pore size of the as-synthesized GO-based adsorbents were favorable for the diffusion and adsorption of the typical pharmaceuticals. The GO reacted with 20 mmol/L DFB (GO-DFB20) exhibited the highest removal for six pharmaceuticals among the prepared adsorbents, and can be separated easily. The adsorption capacities of GO-DFB20 for carbamazepine (CBZ), sulfamethoxazole (SMZ), sulfadiazine (SDZ), ibuprofen (IBP), paracetamol (PCT) and phenacetin (PNT) were 340.5, 428.3, 214.7, 224.3, 350.6 and 316.1 μmol/g, respectively. The adsorption kinetics of PCT on the GO-DFB20 was faster than SMZ. According to the Langmuir fitting, the maximum adsorption capacities of GO-DFB20 for SMZ and PCT were 749.6 and 663.9 μmol/g, respectively. The spent GO-DFB20 was successfully regenerated by methanol with little loss of adsorption capacity in five successive adsorption cycles. This study shows that the porous GO adsorbent has a promising application for the removal of typical pharmaceuticals from water or wastewater.