Search

Abstract: A new, simple, rapid, sensitive, and repeatable reversed-phase HPLC method was developed and validated for the simultaneous determination of tolbutamide, phenacetin and their metabolites in rat liver perfusate. Chlorpropamide was used as an internal standard to ensure the precision and accuracy of this method. Analytes were extracted into diethyl ether using a two-step liquid–liquid extraction. A C18 analytical column and a mobile phase composed of acetonitrile and potassium phosphate buffer were used for the chromatographic separation with UV detection. Limits of detection varied between 20 and 46ng/mL for phenacetin, tolbutamide and their metabolites. The overall extraction recovery for the analytes varied from 65.4% in paracetamol to 88.0% in tolbutamide for concentrations within the expected range of concentrations from previous experimental samples. In terms of precision, the intra- and inter-day variation at three different concentrations in all analytes never exceeded 7.6 and 11.4%, respectively. This method is applicable for the modeling and description of possible pharmacological interactions on rat cytochromes P450 1A2 and 2C6/11 or can be used for in vitro evaluation of both cytochromes 1A2 and 2C. [Copyright &y& Elsevier]

Abstract: Apparent rate constants for the reactions of four selected pharmaceutical compounds (metoprolol, naproxen, amoxicillin, and phenacetin) with chlorine in ultra-pure (UP) water were determined as a function of the pH. It was found that amoxicillin (in the whole pH range 3–12), and naproxen (in the low pH range 2–4) presented high reaction rates, while naproxen (in the pH range 5–9), and phenacetin and metoprolol (in the pH range 2.5–12 for phenacetin, and 3–10 for metoprolol) followed intermediate and slow reaction rates. A mechanism is proposed for the chlorination reaction, which allowed the evaluation of the intrinsic rate constants for the elementary reactions of the ionized and un-ionized species of each selected pharmaceutical with chlorine. An excellent agreement is obtained between experimental and calculated rate constants by this mechanism. The elimination of these substances in several waters (a groundwater, a surface water from a public reservoir, and two effluents from municipal wastewater treatment plants) was also investigated at neutral pH. The efficiency of the chlorination process with respect to the pharmaceuticals elimination and the formation THMs was also established. It is generally observed that the increasing presence of organic and inorganic matter in the water matrices demand more oxidant agent (chlorine), and therefore, less chlorine is available for the oxidation of these compounds. Finally, half-life times and oxidant exposures (CT) required for the removal of 99% of the four pharmaceuticals are also evaluated. These parameters are useful for the establishment of safety chlorine doses in oxidation or disinfection stages of pharmaceuticals in treatment plants. [Copyright &y& Elsevier]

Objectives The effects of nuciferine, a major active aporphine alkaloid from the leaves of Nelumbo nucifera Gaertn, on a cytochrome P450 1A2 (CYP1A2) probe substrate were investigated in vitro and in vivo. Methods Nuciferine and recombinant human CYP1A2 were incubated together to study the impact of nuciferine on CYP1A2 in vitro. Nuciferine was administered orally to Wistar rats at a dose of 20 mg/kg to further estimate the impact of nuciferine on CYP1A2 in vivo. A probe substrate, phenacetin, was used to index the activity of CYP1A2. Key findings The IC50 value for nuciferine was determined to be 2.12 mmol/l. When phenacetin was intravenously coadministered with nuciferine compared with phenacetin alone, the elimination rate constant and total body clearance of phenacetin were decreased by 24.0% ( P < 0.01) and 43.0% ( P < 0.05), respectively. The mean residence time, apparent elimination half-time and area under the plasma concentration-time curve were increased by 22% ( P < 0.005), 26.9% ( P < 0.02) and 74.6% ( P < 0.05), respectively. Similarly, when phenacetin was coadministered orally with nuciferine, the apparent elimination half-time in the nuciferine pretreated group was increased by 16.7% ( P < 0.05) and the elimination rate constant was decreased by 15.4% ( P < 0.05). Conclusions The results suggest that nuciferine inhibited CYP1A2 activity in vitro and caused changes in the pharmacokinetic parameters of phenacetin in vivo. [ABSTRACT FROM AUTHOR]

Buagafuran (BF), derived from α-agarofuran, is a promising anti-anxiety drug in phase I clinical trials. The present study was undertaken to examine the regulation of BF on liver cytochrome P450 (CYP) isoforms in rats. After being administered (4, 16, and 64 mg/kg) by gavage for 7 continuous days, the activities of CYP isoforms were measured by the qualification of six metabolites from CYP probe substrates using LC-MS/MS analysis. The mRNA and protein levels of CYPs were detected by reverse transcription polymerase chain reaction and Western blotting assay, respectively. Using phenacetin and chlorzoxazone as probe drugs, the activities of CYP1A2 and CYP2E1 were monitored in vivo. The result indicated that BF significantly increased the activity and protein levels of CYP1A2 and CYP2E1, while the mRNA levels were elevated to a certain extent. CYP2C6 and CYP2C11 were also slightly induced by BF, but no effect on liver CYP3A was detected in rats. Treatment of BF orally resulted in the decreasing of AUC, MRT and increasing of CL/F of phenacetin as well as production of acetaminophen in rats. The similar pharmacokinetic changes were also observed when using chlorzoxazone as a probe drug. Collectively, BF has inducing potential of liver CYP1A2 and CYP2E1 and may influence the corresponding pharmacokinetics of other drugs. [ABSTRACT FROM AUTHOR]

Abstract: The ozonation of four pharmaceuticals (metoprolol, naproxen, amoxicillin, and phenacetin) in ultra-pure (UP) water was studied in the pH range between 2.5 and 9. The experiments allowed the determination of the apparent rate constants for the reactions between ozone and the selected compounds. The values obtained varied depending on the pH, and ranged between 239 and 1.27×104 M−1 s−1 for metoprolol; 2.62×104 and 2.97×105 M−1 s−1 for naproxen; 2.31×103 and 1.21×107 M−1 s−1 for amoxicillin; and 215 and 1.57×103 M−1 s−1 for phenacetin. Due to the acidic nature of these substances, the degree of dissociation of each pharmaceutical was determined at every pH of work, and the specific rate constants of the neutral and ionic species formed were evaluated. Additionally, the simultaneous ozonation of the pharmaceuticals in different water matrices was carried out by considering a groundwater, a surface water from a public reservoir, and three secondary effluents from municipal wastewater treatment plants. The influence of the operating conditions (initial ozone dose, nature of pharmaceuticals and type of water) on the pharmaceuticals elimination efficiency was established, and a kinetic model was proposed for the evaluation of the partial contribution to the global oxidation of both, the direct ozonation reaction and the radical pathway. [Copyright &y& Elsevier]

The preparation of a methacrylate polymer molecularly imprinted (MIP) with paracetamol (APAP) was performed. After extraction of the APAP template molecule, the MIPs were incorporated into a graphite-polyurethane (GPU) matrix, and the resulting composites were used to prepare modified electrodes intended to be used in APAP determination. The best results were found using a 2.5% MIP in the GPU electrode and a 500-µm MIP particle size. This electrode was used in the determination of APAP in pharmaceutical formulations, reaching a 6.7 × 10-8 mol L-1 limit of detection. The 2.5% MIP-GPU-modified electrode showed better sensitivity than the nonimprinted methacrylate GPU-modified electrodes. Interference of phenacetin in the APAP response decreased remarkably when the proposed electrode was used. [ABSTRACT FROM AUTHOR]

A highly sensitive liquid chromatographic-atmospheric pressure chemical ionization-tandem mass spectrometric method is developed to quantitate phenacetin and its metabolite paracetamol in rabbit plasma. The analytes and internal standard oxazepam are extracted from plasma by liquid–liquid extraction using ethyl acetate, and separated on a Zorbax SB-C18 column (2.1 mm × 150 mm, 5 μm) using acetonitrile–0.1% formic acid in water (40:60 v/ v) at a flow of 0.4 mL min−1. Detection is carried out by multiple reaction monitoring on a ion-trap LC-MS-MS system with an atmospheric pressure chemical ionization interface. The assay is linear over the range 4–1,600 ng mL−1 for phenacetin and 3–2,000 ng mL−1 for paracetamol, with a lower limit of quantitation of 4 ng mL−1 for phenacetin and 3 ng mL−1 for paracetamol. Intra- and inter-day precision are less than 7.1% and the accuracy are in the range 97.3–103.5%. The validated method is successfully used to analyze the drug in samples of rabbit plasma for pharmacokinetic study. [ABSTRACT FROM AUTHOR]

A recent focus was to investigate whether antofloxacin, an 8-NH2 derivative of levofloxacin, inhibited cytochrome P450 (CYP) 1A2 activity in rats. Phenacetin, the representative substrate of CYP1A2, was used as the model drug to evaluate the activity of CYP1A2. In an in vivo study, an oral single dose of antofloxacin (20 mg kg-1) did not affect the pharmacokinetic behaviour of phenacetin, but a multidose (20 mg kg-1 twice daily for 7.5 days) significantly increased phenacetin's area under the curve (AUC). In an in vitro study, only when pre-incubated with β-nicotinamide adenine dinucleotide phosphate, a reduced form (NADPH) system in rat liver microsomes, did antofloxacin inhibit phenacetin O-deethylation. The inhibition was NADPH-, pre-incubation time-, and antofloxacin concentration-dependent. A physiologically based pharmacokinetic model with mechanism-based inhibition was successfully developed for predicting the interaction between antofloxacin and phenacetin in vivo from the in vitro data. The simulated AUC was 1.4-fold of the control, which was near the observed value of 1.6-fold. From the results, it can be concluded that the inhibition of CYP1A2 by antofloxacin is mechanism-based. [ABSTRACT FROM AUTHOR]

The solubility of phenacetin, salicylic acid, and probenecid in ethanol–water and ethanol–ethyl acetate mixtures at several temperatures (15–40°C) was measured. The solubility profiles are related to medium polarity changes. The apparent thermodynamic magnitudes and enthalpy–entropy relationships are related to the cosolvent action. Salicylic acid and probenecid show a single peak against the solubility parameter δ1 of both solvent mixtures, at 40% (δ1 = 21.70 MPa1/2) and 30% (δ1 = 20.91 MPa1/2) ethanol in ethyl acetate, respectively. Phenacetin displays two peaks at 60% ethanol in ethyl acetate (23.30 MPa1/2) and 90% ethanol in water (δ1 = 28.64 MPa1/2). The apparent enthalpies of solution display a maximum at 30% (phenacetin and salicylic acid) and 40% (probenecid) ethanol in water, respectively. Two different mechanisms, entropy at low ethanol ratios, and enthalpy at high ethanol ratios control the solubility enhancement in the aqueous mixture. In the nonaqueous mixture (ethanol–ethyl acetate) enthalpy is the driving force throughout the whole solvent composition for salicylic acid and phenacetin. For probenecid, the dominant mechanism shifts from entropy to enthalpy as the ethanol in ethyl acetate concentration increases. The enthalpy–entropy compensation plots corroborate the different mechanisms involved in the solubility enhancement by cosolvents. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:1129–1135, 2009 [ABSTRACT FROM AUTHOR]

The article discusses ways to model pharmaceuticals in cases which only limited experimental data is available. The authors propose a methodology to model the phase equilibria of complex chemicals, including pharmaceuticals, using equation of state (EoS) models. Pure fluid parameters are estimated using limited experimental or predicted data on sublimation pressures and liquid densities, the authors state. Other topics include modeling the solubility of the pharmaceuticals which include acetanilide, phenacetin, and paracetamol.

Abstract: A sensitive and specific liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method has been developed and validated for the simultaneous quantification of tetrahydropalmatine, protopine and palmatine in rat plasma using phenacetin as the internal standard (IS). Two hundred microliters plasma samples were extracted by dichloromethane under a strong basic condition. The analytes were separated by a C18 column and detected with a single quadrupole mass spectrometer. The used mobile phase was acetonitrile–water (40:60, v/v) containing 5mM ammonium acetate and 0.2% glacial acetic acid. Detection was carried out by positive electrospray ionization in selected ion reaction (SIR) mode at m/z 356.6 for tetrahydropalmatine, 354.6 for protopine, 352.6 for palmatine and 180.4 for the IS, respectively. The method was validated over the concentration range of 1.00–500ngmL−1 and the lower limit of quantification (LLOQ) was 1.00ngmL−1 for all three analytes. The intra- and inter-day precision values were less than 9% relative standard deviation (R.S.D.), and the relative error ranged from −7.4 to 4.8%. The extraction recoveries were on average 91.42% for tetrahydropalmatine, 84.75% for protopine, 57.26% for palmatine, and 83.18% for IS. The validated method was successfully applied to a pharmacokinetic study of tetrahydropalmatine, protopine and palmatine in rats after oral administration of Rhizoma Corydalis Decumbentis extract. [Copyright &y& Elsevier]

Recent guidance from the US Food and Drug Administration (USFDA) has advocated testing of time-dependent inhibition of cytochrome P450 (CYP), which can be addressed by performing IC50 shift as well as KI/kinact determinations. Direct (IC50, Ki) and time-dependent inhibition (IC50 shift, KI/kinact) assays were validated in human liver microsomes with liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis for the following enzyme/substrate/inhibitor combinations: CYP1A2/phenacetin/alpha-naphthoflavone/furafylline, CYP2C8/amodiaquine/montelukast/gemfibrozil-1-O-β-glucuronide, CYP2C9/diclofenac/sulfaphenazole/tienilic acid, CYP2C19/S-mephenytoin/S-benzylnirvanol/S-fluoxetine, CYP2D6/dextromethorphan/quinidine/paroxetine, and CYP3A4/midazolam/testosterone/ketoconazole/azamulin/verapamil/diltiazem. IC50 shift assays were performed with two pre-incubation time points (10 and 30 min) to facilitate kinact assay design. Data obtained show good agreement with literature values. For rapid acting inhibitors, such as azamulin/CYP3A4 and tienilic acid/CYP2C9, the IC50 shifts were similar at both time points suggesting a short maximum pre-incubation time with closely spaced time points for the KI/kinact assay. Slow acting inhibitors (such as verapamil/CYP3A4 or S-fluoxetine/CYP2C19) showed an increase in IC50 shift between 10 and 30 min suggesting a longer maximum pre-incubation time with wider spacing of time points for KI/kinact. The two-time point IC50 shift experiment proved to be an excellent method for the selection of appropriate KI/kinact assay parameters and is suitable for the routine analysis of P450 inhibition by drug candidates. [ABSTRACT FROM AUTHOR]

Abstract: This study investigated the effect of berberine on the early phase of hepatocarcinogenesis stimulated by diethylnitrosamine (DEN, 150mg/kg, 4weeks) plus phenobarbital (PB, 75mg/kg, 7days) in rats. The expressions of proliferating cell nuclear antigen (PCNA) and inducible nitric oxide synthase (iNOS) were evaluated by immunohistochemistry. The activities of CYP isoenzymes were analyzed using different probe drugs including chlorzoxazone (CYP2E1) and phenacetin (CYP1A2) by high-performance liquid chromatography (HPLC) in vivo or in vitro. Results showed that the expressions of PCNA and iNOS were induced by DEN plus PB in liver tissues. Oral administration of berberine (50mg/kg) inhibited the hepatocyte proliferation and iNOS expression, decreased cytochrome P450 content, inhibited activities of CYP2E1 and CYP1A2 in DEN-plus-PB-treated rats in vivo. Moreover, berberine (10, 50 and 100μM) inhibited the activities of CYP2E1 and CYP1A2 in microsomes isolated from DEN-plus-PB-treated rats in vitro, suggesting that anti-hepatocarcinogenetic potential of berberine might be due to inhibiting oxidative metabolic activities of CYP 2E1 and CYP1A2, and decreasing NO production in rats. [Copyright &y& Elsevier]

Abstract: In the paper, the poly(d,l-lactide) (PDLLA) and poly(ethylene glycol)-co-poly(d,l-lactide) (PELA) fibers with and without paracetanol drug loading were prepared with an electrospinning method. The morphology of the fibers was observed by scanning electronic microscope (SEM). Their glass transition temperatures (T g) were measured with differential scanning calorimetry (DSC). The water contact angle (CA) measurement was also performed to characterize surface properties of fibers. At 37°C in a PBS buffer solution (pH 7.4), in vitro matrix degradation profiles of these fibers were characterized by measuring their weight loss, the molecular weight decrease, and their morphology change. The result showed that the effects of fiber diameter and porosities on the degradation of the electrospun scaffolds might exceed the effects of the molecular weight and the PEG contents, which was different from the polymeric microspheres degradation. In vitro paracetanol release profiles were also investigated in the same condition. The result showed that the drug burst release behaviour was mainly related with the drug–polymer compatibility and the followed sustained release phase depended on polymer degradation. [Copyright &y& Elsevier]

Aim: CYP1A2, a constitutive enzyme expressed in the liver, is among the phase I enzymes responsible for polycyclic aromatic hydrocarbons metabolism. Phenacetin O-de-ethylation is a marker for CYP1A2 activity. This study investiagtes the metabolism of phenacetin in patients with hepatocellular carcinoma (HCC). Methods: The phenacetin test was performed in 56 normal subjects and 92 HCC patients. The test was repeated in HCC patients after treatment with transcatheter arterial chemoembolization (TACE). The recovery of phenacetin’s urinary metabolites was studies in 12 normal subjects and 14 patients with HCC. Results: Compared with normal controls, the recovery of phenacetin O-de-ethylated metabolites decreased by 42·5% ( P < 0·01) in patients with HCC and diminished further after TACE ( P < 0·05). The ratio of plasma total paracetamol to phenacetin was much lower than in normal controls ( P < 0·01) and was reduced by 40·7% more after TACE ( P < 0·05). Conclusions: The metabolism of phenacetin is impaired in patient with HCC. TACE damages the activity of CYP1A2. The phenacetin test can be used to predict effect of TACE on liver function in HCC patients. [ABSTRACT FROM AUTHOR]

Abstract: The present study developed a validate and precise reversed-phase high performance liquid chromatography (HPLC) method for the determination of thalidomide (T) in plasma, to quantify T in patients affected by hepatocellular carcinoma. Twelve male subjects aging from 62 to 82 years and weighting 66–88kg, were orally administered with single dose of T (200mg/BW). Two ml of stabilizer-solution (CH3OH/CH3CN, 1/1 (v/v)+CH3COOH 2%) were added to 1ml of human plasma and stoked to −80°C until analyses. This moisture (1.38μl) was added with 20μl of CF3COOH and 100μl of phthalimide (IS) 1.75μg/ml, vortexed and centrifuged. Surnatant (800μl) was dried under vacuum at room temperature, added with 50μl of appropriate solution and injected onto HPLC. T and IS were detected at UV wavelength of 220nm with a run time of 10min. Mobile phase was 10mM pH 5.5NH4 +CH3COO−/CH3CN, 75/25 (v/v) buffer at flow rate of 1.5ml/min. Inter-day and intra-day variation coefficient was <10% with an error of accuracy <10%. The present detection method was able to quantify T to every withdrawal time period (LOD 0.05μg/ml). The IS used in the present study had the same wavelength maximum absorption of T, differently from early UV detection methods reported in literature where phenacetin was used. Pharmacokinetic parameters belonging from the present study are not significantly different from those calculated in previously studies performed in human health subjects and patients affected by other pathology. [Copyright &y& Elsevier]

The cytochrome P450 enzyme CYP1A2 is crucial for the metabolism of many drugs, for example, tizanidine. As the effects of several non-steroidal anti-inflammatory drugs (NSAID) and female sex steroids on CYP1A2 activity in vitro are unknown, their effects on phenacetin O-deethylation were studied and compared with the effects of model inhibitors in human liver microsomes, followed by prediction of their interaction potential with tizanidine in vivo. In vitro, fluvoxamine, tolfenamic acid, mefenamic acid and rofecoxib potently inhibited CYP1A2 [the 50% inhibitory concentration (IC50) < 10 µM]. Ethinyloestradiol, celecoxib, desogestrel and zolmitriptan were moderate (IC50 20–200 µM), and etodolac, ciprofloxacin, etoricoxib and gestodene weak inhibitors of CYP1A2 (IC50 > 200 µM). At 100 µM, the other tested NSAIDs and steroids inhibited CYP1A2 less than 35%. Pre-incubation increased the inhibitory effects of rofecoxib, progesterone and desogestrel. Using the free portal plasma inhibitor concentration and the competitive inhibition model, the effect of fluvoxamine and the lack of effects of tolfenamic acid and celecoxib on tizanidine pharmacokinetics in human beings were well predicted. However, the effects of ciprofloxacin, rofecoxib and oral contraceptives were greatly underestimated even when the predictions were based on their total portal plasma concentration. Besides rofecoxib, and possibly mefenamic acid, other NSAIDs were predicted not to significantly inhibit CYP1A2 in human beings. The type of enzyme inhibition, particularly metabolism-dependent inhibition, free inhibitor concentration and accumulation of the inhibitor into the hepatocytes should be considered in extrapolations of in vitro results to human beings. [ABSTRACT FROM AUTHOR]

We recently discovered that rofecoxib is a potent mechanism-based inhibitor of CYP1A2. The effect of the widely used cyclo-oxygenase-2 selective non-steroidal anti-inflammatory drug celecoxib on CYP1A2 activity has not been reported. The effect of celecoxib on CYP1A2 activity (phenacetin O-deethylation) was first studied in vitro using human liver microsomes. This was followed by a randomized, placebo-controlled, cross-over study in which 12 healthy volunteers were given celecoxib (200 mg twice daily) or placebo for 4 days. On day 3, a caffeine test was performed. On day 4, the subjects ingested 2 mg tizanidine. Plasma samples for the measurement of the concentrations of tizanidine, its metabolites and celecoxib were collected up to 24 h post-administration. Pharmacodynamic variables (e.g. blood pressure, subjective drowsiness and drug effect) were recorded up to 12 h post-adm. Celecoxib was found to be a moderately potent competitive inhibitor of CYP1A2 in vitro with a Ki (inhibitor constant) of 25.4 μ M. However, in vivo, celecoxib did not affect the caffeine test, or the peak concentration, time to peak concentration, area under the concentration-time curve or half-life of tizanidine. The pharmacodynamic variables of tizanidine also remained unchanged. Unlike rofecoxib, celecoxib does not clinically to significantly inhibit CYP1A2. The lack of significant in vivo inhibition of CYP1A2 can be correctly predicted on the basis of in vitro Ki data and the free peripheral or portal plasma concentration of celecoxib. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To determine whether duloxetine is a substrate, inhibitor or inducer of cytochrome P450 (CYP) 1A2 enzyme, using in vitro and in vivo studies in humans. METHODS: Human liver microsomes or cells with expressed CYP enzymes and specific CYP inhibitors were used to identify which CYP enzymes catalyse the initial oxidation steps in the metabolism of duloxetine. The potential of duloxetine to inhibit CYP1A2 activity was determined using incubations with human liver microsomes and phenacetin, the CYP1A2 substrate. The potential for duloxetine to induce CYP1A2 activity was determined using human primary hepatocytes treated with duloxetine for 72 hours. Studies in humans were conducted using fluvoxamine, a potent CYP1A2 inhibitor, and theophylline, a CYP1A2 substrate, as probes. The subjects were healthy men and women aged 18–65 years. Single-dose duloxetine was administered either intravenously as a 10-mg infusion over 30 minutes or orally as a 60-mg dose in the presence or absence of steady-state fluvoxamine (100 mg orally once daily). Single-dose theophylline was given as 30-minute intravenous infusions of aminophylline 250 mg in the presence or absence of steady-state duloxetine (60 mg orally twice daily). Plasma concentrations of duloxetine, its metabolites and theophylline were determined using liquid chromatography with tandem mass spectrometry. Pharmacokinetic parameters were estimated using noncompartmental methods and evaluated using mixed-effects ANOVA. Safety measurements included vital signs, clinical laboratory tests, a physical examination, ECG readings and adverse event reports. RESULTS: The in vitro results indicated that duloxetine is metabolized by CYP1A2; however, duloxetine was predicted not to be an inhibitor or inducer of CYP1A2 in humans. Following oral administration in the presence of fluvoxamine, the duloxetine area under the plasma concentration-time curve from time zero to infinity (AUC∞) and the maximum plasma drug concentration (Cmax) significantly increased by 460% (90% CI 359, 584) and 141% (90% CI 93, 200), respectively. In the presence of fluvoxamine, the oral bioavailability of duloxetine increased from 42.8% to 81.9%. In the presence of duloxetine, the theophylline AUC∞ and Cmax increased by only 13% (90% CI 7, 18) and 7% (90% CI 2, 14), respectively. Coadministration of duloxetine with fluvoxamine or theophylline did not result in any clinically important safety concerns, and these combinations were generally well tolerated. CONCLUSION: Duloxetine is metabolized primarily by CYP1A2; therefore, coadministration of duloxetine with potent CYP1A2 inhibitors should be avoided. Duloxetine does not seem to be a clinically significant inhibitor or inducer of CYP1A2; therefore, dose adjustment of CYP1A2 substrates may not be necessary when they are coadministered with duloxetine. [ABSTRACT FROM AUTHOR]

Abstract: In this study, wild-type human CYP1A2 without the conventional N-terminal modification (second codon GCT) or the truncation of the N-terminal hydrophobic region was functionally expressed in Escherichia coli. Its enzymatic properties were compared with N-terminally modified CYP1A2. Although modified CYP1A2 is almost all high-spin, some wild-type CYP1A2 shifted to low-spin. Spectral binding titrations with several ligands could be performed with wild-type enzyme, but not with modified enzyme. Kinetic parameters for several substrates were similar for the two CYP1A2 enzymes. However, the oxidation rates of phenacetin by modified enzyme were ∼2-fold higher than those by wild-type enzyme. The intermolecular isotope effects were ∼2 for phenacetin O-deethylation catalyzed by both enzymes. However, the wild-type enzyme, but not the modified enzyme, increased C-hydroxylation when O-deethylation rates were lowered by deuterium substitution. Molecular switching indicates that phenacetin rotates within the active site of wild-type enzyme and suggests a looser conformation in the active site of the wild-type enzyme than of the modified enzyme. These results reveal that the overall enzymatic properties of wild-type CYP1A2 enzyme are quite similar to those of modified CYP1A2, although its active site environment seems to differ from that of the modified enzyme. [Copyright &y& Elsevier]

Abstract: Quantitative infrared (IR) and Raman spectroscopic approach for determination of phenacetin (Phen) and salophen (Salo) in binary solid mixtures with caffeine: phenacetin/caffeine (System 1) and salophen/caffeine (System 2) is presented. Absorbance ratios of 746cm−1 or 721cm−1 peaks (characteristic for each of determined compounds in the Systems 1 and 2) to 1509cm−1 and 1616cm−1 (attributed to Phen and Salo, respectively) were used. The IR spectroscopy gives confidence of 98.9% (System 1) and 98.3% (System 2), while the Raman spectroscopic data are with slightly higher confidence of 99.1% for both systems. The limits of detection for the compounds studied were 0.013 and 0.012mole fraction for IR and Raman methods, respectively. Solid-state linear dichroic infrared (IR-LD) spectral analysis of solid mixtures was carried out with a view to obtaining experimental IR spectroscopic assignment of the characteristic IR bands of both determined compounds. The orientation technique as a nematic liquid crystal suspension was used, combined with the so-called reducing-difference procedure for polarized spectra interpretation. The possibility for obtaining supramolecular stereo structural information for Phen and Salo by comparing spectroscopic and crystallographic data has also been shown. An independent high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) analysis was performed for comparison and validation of vibrational spectroscopy data. Applications to 10 tablets of commercial products APC and Sedalgin are given. [Copyright &y& Elsevier]

Our aim was to correlate the in vitro and in vivo CYP1A2 inhibition potential of tolfenamic acid, an NSAID highly (99.7%) bound to plasma proteins, to study the significance of protein binding of inhibitor in metabolic drug interactions. The effect of tolfenamic acid on CYP1A2 (phenacetin O-deethylation) was studied using human liver microsomes, with and without albumin (0–10 mg/ml). In a randomized, crossover study, 10 volunteers took 200 mg tolfenamic acid or placebo t.i.d. for 3 days. On day 2, a caffeine test was performed. On day 3, each ingested 4 mg of the CYP1A2 substrate tizanidine. Plasma tizanidine, its metabolites (M) and tolfenamic acid, and pharmacodynamic variables were measured. Tolfenamic acid strongly inhibited phenacetin-O-deethylation in vitro (IC50 1.8 μM without albumin). Albumin decreased its inhibitory effect in a concentration-dependent manner; the IC50 exceeded 100 μM with 10 mg/ml of albumin. Tolfenamic acid had no effect on the area under the concentration-time curve $${\left( {{\text{AUC}}_{{0 - \infty }} } \right)}$$ , peak concentration, time of peak concentration or half-life of tizanidine or M-3; only the $${\text{AUC}}_{{0 - \infty }} $$ of secondary metabolite M-4 was slightly decreased (13%, P = 0.004). The caffeine test and the pharmacodynamic effects of tizanidine were unchanged. Tolfenamic acid potently inhibits CYP1A2 in vitro when studied without albumin, but not in vivo. This apparent discrepancy is due to the high protein binding of tolfenamic acid. To avoid overestimation of the interaction potential, the inhibitory effect of highly albumin-bound compounds should also be studied in vitro with albumin, or their exact unbound plasma concentration should be used in predictions. [ABSTRACT FROM AUTHOR]

Although the mechanism of action of acetaminophen (AAP) is not fully understood, some studies suggest that AAP and phenacetin (PHE) are selective cyclooxygenase (COX)-3 inhibitors. To examine the participation of COX-3 in memory formation, water maze performance was studied in mice treated with AAP, PHE or other COX inhibitors. Mice received intraperitoneal injections of drugs immediately after each training session. Administration of high-dose AAP [302.3 mg/kg (IC50 for COX-2)] or PHE [179.2 mg/kg (IC50 for COX-2)] and of non-specific (indomethacin: 20 mg/kg) or specific COX-2 (NS-398:10 mg/kg) inhibitor impaired the performance in hidden platform (HP) not visible platform (VP) tasks, whereas tow-dose (15.1 mg/kg) AAP facilitated performance in HP and VP tasks. The facilitation of performance by low-dose AAP was reversed by co-administration with a 5-HT½ receptor antagonist (methysergide: 0.47 mg/kg). The middle-dose [69.5 mg/kg (IC50 for COX-3)] of AAP, the PHE [17.9 mg/kg (IC50 for COX-3)] and a specific COX-1 inhibitor (piroxicam: 10-20 mg/kg) did not influence performance in either task. These results suggest that the memory impairment by high-dose AAP and PHE and facilitation of performance by tow-dose AAP could involve endogenous COX-2 and serotonergic neuronal activity, but not COX-3, respectively. [ABSTRACT FROM AUTHOR]

In contrast to nonsteroidal anti-inflammatory drugs (NSAIDs), the nonopioid analgesics phenacetin, acetaminophen and dipyrone exhibit weak anti-inflammatory properties. An explanation for this difference in pharmacologic activity was provided by the recent discovery of a new cyclooxygenase isoform, cyclooxygenase (COX)-3, that is reported to be inhibited by phenacetin, acetaminophen and dipyrone. However, COX-3 was found to be a spliced variant of COX-1 and renamed COX-1b. Although recent studies provide evidence for the existence of this new COX isoform, it is uncertain whether this COX-3 (COX-1b) isoform, or putative acetaminophen-sensitive pathway, plays a role in the generation of vasoactive prostaglandins. NSAIDs increase systemic blood pressure by inhibiting the formation of vasodilator prostanoids. Angiotensin II, norepinephrine and other vasoconstrictor agents have been reported to release prostaglandins. It is possible that this acetaminophen-sensitive pathway also modulates pressor responses to these vasoconstrictor agents. Therefore, the purpose of the present study was to determine whether this acetaminophen-sensitive pathway plays a role in the generation of vasoactive products of arachidonic acid or in the modulation of vasoconstrictor responses in the pulmonary and systemic vascular bed of the intact-chest rat. In the present study, the nonopioid analgesics did not attenuate changes in pulmonary or systemic arterial pressure in response to injections of the prostanoid precursor, arachidonic acid, to the thromboxane A2 mimic, U46619, or to angiotensin II or norepinephrine. The results of the present study do not provide evidence in support of a role of a functional COX-3 (COX-1b) isoform, or an acetaminophen-sensitive pathway, in the generation of vasoactive prostanoids or in the modulation of responses to vasoconstrictor hormones in the intact-chest rat. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: A sensitive and accurate HPLC-UV method for the quantification of fluconazole (FLA) level in human plasma has been developed. The sample was prepared by one-step liquid–liquid extraction (LLE) of FLA from plasma using dichloromethane. Phenacetin was used as the internal standard. The chromatographic retention times of FLA and phenacetin were 4.6 and 8.3min, respectively. The lower limit of quantitation (LLOQ) was 0.05μg/mL, and no interferences were detected in the chromatograms. The devised HPLC-UV method was validated by evaluating its intra- and inter-day precisions and accuracies in a linear concentration range between 0.05 and 10.00μg/mL. The devised method was successfully applied to a bioequivalence studies involving the oral administration of a single 150mg FLA tablet and 3×50mg FLA capsules in healthy Korean male volunteers. [Copyright &y& Elsevier]

Abstract: Substrate inhibition assays for five of the major CYP enzymes (phenacetin for CYP1A2, diclofenac for CYP2C9, (S)-mephenytoin for CYP2C19, dextromethorphan for CYP2D6 and midazolam and testosterone for CYP3A4) in human liver microsomes were developed. Fully automated incubations were conducted in a 96-well format under optimized enzyme kinetic conditions. Metabolites of probe substrates were analyzed with rapid LC–MS/MS methods. The assays were fully validated following the procedure for validating bioanalytical methods recommended by regulatory agencies. Quality control samples and a positive control CYP inhibitor were included in each assay. The IC50 values determined for typical CYP inhibitors were reproducible and consistent with those reported in the literature. The high quality and throughput of these assays make them ideally suited for providing information for decision making in late drug discovery and early development and for providing labeling input for new drug registrations. [Copyright &y& Elsevier]

Introduction: The absorption process is an essential stage that determines the bioavailability of xenobiotics during extravascular administration. The intensity of absorption depends on numerous factors not necessarily related to the substance and its formulation, and also on biotransformation and active transport processes. Methanol and its water solutions facilitate the animal dosage of several drugs, inhibitors and other xenobiotics in a soluble form while these compounds are not soluble in water. However, during administration of small amounts of drugs, the effect of vehiculum on drug fate in the body becomes also evident. Material and Methods: The study was performed on male Wistar rats cmd(WI)WU. Phenacetin was given per os in a single dose of 100 mg/kg b.w. Various procedures of phenacetin administration were tested, including solubilization in methanol and/or suspension in 1% water solution of carboxymethylcellulose. A three-hour pretreatment with 75% methanol solution before phenacetin administration was also applied. Serum concentrations of phenacetin and acetaminophen, its metabolite, were determined by HPLC method with solid phase extraction. Results and Conclusions: The presented results indicate that methanol decreases blood acetaminophen concentrations and phenacetin bioavailability. These changes appeared immediately after methanol exposure, but during the last four weeks of the experiment, when methanol was not in use, and only phenacetin in 1% carboxymethylcellulose was administered to rats, the decrease in phenacetin biotransformation and bioavailability was maintained. [ABSTRACT FROM AUTHOR]

Introduction: Exposure to some chemical agents is known to cause alteration in lipid metabolism and oxidative stress. The aim of the experiment reported in this paper was to reveal the effects of gasoline vapours on serum lipid profile and oxidative stress in rats. Material and Methods: The study was performed on Wistar male and female rats. The animals were exposed to gasoline vapours generated from direct evaporation of liquid gasoline at concentration 17.8±2.6 cm3 hr-1kg-1m-3 day-1, 4 h/day for 64 days. Results: Compared with the control groups, there was a significant decrease in the activity of superoxide dismutase and an increase in the extent of lipid peroxidation (malondialdehyde) in the liver tissue of the exposed rats. Gasoline vapours enhanced the level of serum cholesterol, triglyceride, LDL, and VLDL as well as decreased the concentration of HDL. Changes in serum lipid profile were more pronounced in female than in male rats. Conclusions: Gasoline vapours induced proatherogenic changes in serum lipid profile and signs of oxidative stress in the liver of the exposed rats. Based on these results, it may be inferred that the adverse effects associated with exposure to gasoline vapours are gender-dependent, with females being more vulnerable than males. [ABSTRACT FROM AUTHOR]

Background: An ad hoc peer-review committee was jointly appointed by Drug Authorities and Industry in Germany, Austria and Switzerland in 1999/2000 to review the evidence for a causal relation between phenacetin-free analgesics and nephropathy. The committee found the evidence as inconclusive and requested a new case-control study of adequate design. Methods: We performed a population-based case-control study with incident cases of end-stage renal disease (ESRD) under the age of 50 years and four age and sex-matched neighborhood controls in 170 dialysis centers (153 in Germany, and 17 in Austria) from January 1, 2001 to December 31, 2004. Data on lifetime medical history, risk factors, treatment, job exposure and intake of analgesics were obtained in a standardized face-to-face interview using memory aids to enhance accuracy. Study design, study performance, analysis plan, and study report were approved by an independent international advisory committee and by the Drug Authorities involved. Unconditional logistic regression analyses were performed. Results: The analysis included 907 cases and 3,622 controls who had never used phenacetincontaining analgesics in their lifetime. The use of high cumulative lifetime dose (3rd tertile) of analgesics in the period up to five years before dialysis was not associated with later ESRD. Adjusted odds ratios with 95% confidence intervals were 0.8 (0.7 - 1.0) and 1.0 (0.8 - 1.3) for evercompared with no or low use and high use compared with low use, respectively. The same results were found for all analgesics and for mono-, and combination preparations with and without caffeine. No increased risk was shown in analyses stratifying for dose and duration. Dose-response analyses showed that analgesic use was not associated with an increased risk for ESRD up to 3.5 kg cumulative lifetime dose (98 % of the cases with ESRD). While the large subgroup of users with a lifetime dose up to 0.5 kg (278 cases and 1365 controls) showed a significantly decreased risk, a tiny subgroup of extreme users with over 3.5 kg lifetime use (19 cases and 11 controls) showed a significant risk increase. The detailed evaluation of 22 cases and 19 controls with over 2.5 kg lifetime use recommended by the regulatory advisors showed an impressive excess of other conditions than analgesics triggering the evolution of ESRD in cases compared with controls. Conclusion: We found no clinically meaningful evidence for an increased risk of ESRD associated with use of phenacetin-free analgesics in single or combined formulation. The apparent risk increase shown in a small subgroup with extreme lifetime dose of analgesics is most likely an indirect, non-causal association. This hypothesis, however, cannot be confirmed or refuted within our case-control study. Overall, our results lend support to the mounting evidence that phenacetin-free analgesics do not induce ESRD and that the notion of "analgesic nephropathy" needs to be re-evaluated. [ABSTRACT FROM AUTHOR]

Background: Use of phenacetin and other analgesic and non-steroidal anti-inflammatory drugs (NSAIDs) potentially influences bladder cancer incidence, but epidemiologic evidence is limited. Methods: We analyzed data from 376 incident bladder cancer cases and 463 controls from a population-based case-control study in New Hampshire on whom regular use of analgesic drugs and NSAIDs was obtained. Odds ratios and 95% confidence intervals were computed using logistic regression with adjustment for potentially confounding factors. Separate models by tumor stage, grade and TP53 status were conducted. Results: We found an elevated odds ratio (OR) associated with reported use of phenacetincontaining medications, especially with longer duration of use (OR >8 years = 3.00, 95% confidence interval (CI) = 1.4-6.5). In contrast, use of paracetamol did not relate overall to risk of bladder cancer. We also found that regular use of any NSAID was associated with a statistically significant decrease in bladder cancer risk (OR = 0.6, 95% CI = 0.4-0.9), and specifically use of aspirin. Further, the association with NSAID use was largely among invasive, high grade and TP53 positive tumors. Conclusion: While these agents have been investigated in several studies, a number of questions remain regarding the effects of analgesic and NSAID use on risk of bladder cancer. [ABSTRACT FROM AUTHOR]

A simple and rapid HPLC method using phenacetin (PHN) as internal standard has been developed for simultaneous determination of acetaminophen, caffeine, and chlorphenamine maleate in the product compound paracetamol and chlorphenamine maleate granules. Separation and quantitation were achieved on a 250 mm × 4.6 mm, 5 μm particle, C18 column. The mobile phase was methanol 0.05 mol L−1 aqueous KH2PO4 solution, 45:55 ( v/ v), containing 0.1% triethylamine and adjusted to pH 3.6 by addition of phosphoric acid; the flow rate was 1.0 mL min−1. Detection of all compounds was by UV absorbance at 260 nm and elution of the analytes was achieved in less than 12 min. The linearity, accuracy, and precision of the method were acceptable to good over the concentration ranges 6.4–153.6 μg mL−1 for acetaminophen, 5.0–120.0 μg mL−1 for caffeine, and 9.6–230.4 μg mL−1 for chlorphenamine maleate. [ABSTRACT FROM AUTHOR]

Temperature dependencies of saturated vapor pressure for the monoclinic modification of paracetamol (acetaminophen), acetanilide, and phenacetin (acetophenetidin) were measured and thermodynamic functions of sublimation calculated (paracetamol: ΔGsub298 = 60.0 kJ/mol; ΔHsub298 = 117.9 ± 0.7 kJ/mol; ΔSsub298 = 190 ± 2 J/mol · K; acetanilide: ΔGsub298 = 40.5 kJ/mol; ΔHsub298 = 99.8 ± 0.8 kJ/mol; ΔSsub298 = 197 ± 2 J/mol · K; phenacetin: ΔGsub298 = 52.3 kJ/mol; ΔHsub298 = 121.8 ± 0.7 kJ/mol; ΔSsub298 = 226 ± 2 J/mol · K). Analysis of packing energies based on geometry optimization of molecules in the crystal lattices using diffraction data and the program Dmol3 was carried out. Parameters analyzed were: (a) energetic contribution of van der Waals forces and hydrogen bonding to the total packing energy; (b) contributions of fragments of the molecules to the packing energy. The fraction of hydrogen bond energy in the packing energy increases as: phenacetin (17.5%) < acetanilide (20.4%) < paracetamol (34.0%). Enthalpies of evaporation were estimated from enthalpies of sublimation and fusion. Activity coefficients of the drugs in n-octanol were calculated from cryoscopic data and by estimation of dilution enthalpy obtained from solubility and calorimetric experiments (for infinite dissolution). Solubility temperature dependencies in n-octanol and n-hexane were measured. The thermodynamic functions of solubility and solvation processes were deduced. Specific and nonspecific solvation terms were distinguished using the transfer from the “inert” n-hexane to the other solvents. The transfer of the molecules from water to n-octanol is enthalpy driven for paracetamol; for acetanilide and phenacetin, entropy driven. © 2006 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 95:2158–2169, 2006 [ABSTRACT FROM AUTHOR]

Abstract: The solubility enhancement produced by two binary mixtures with a common cosolvent (ethanol–water and ethyl acetate–ethanol) was studied against the solubility parameter of the mixtures (δ 1) to characterize different types of solubility profiles. Benzocaine, salicylic acid and acetanilide show a single peak in the least polar mixture (ethanol–ethyl acetate) at δ 1 =22.59, 21.70 and 20.91MPa1/2, respectively. Phenacetin displays two solubility maxima, at δ 1 =25.71 (ethanol–water) and at δ 1 =23.30 (ethyl acetate–ethanol). Acetanilide shows an inflexion point in ethanol–water instead of a peak, and the sign of the slope does not vary when changing the cosolvent. The solubility profiles were compared to those obtained in dioxane–water, having a solubility parameter range similar to that covered with the common cosolvent system. All the drugs reach a maximum at about 90% dioxane (δ 1 =23MPa1/2). A modification of the extended Hildebrand method is applicable for curves with a single maximum whereas a model including the Hildebrand solubility parameter δ 1 and the acidic partial solubility parameter δ 1a is required to calculate more complex solubility profiles (with inflexion point or two maxima). A single equation was able to fit the solubility curves of all drugs in the common cosolvent system. The polarity of the drug is related to the shape of the solubility profile against the solubility parameter δ 1 of the solvent mixtures. The drugs with solubility parameters below 24MPa1/2 display a single peak in ethanol–ethyl acetate. The drugs with δ 2 values above 25MPa1/2 show two maxima, one in each solvent mixture (ethanol–water and ethanol–ethyl acetate). The position of the maximum in ethanol–ethyl acetate shifts to larger polarity values (higher δ 1 values) as the solubility parameter of the drug δ 2 increases. [Copyright &y& Elsevier]

Abstract: The application of the second most popular artificial neural networks (ANNs), namely, the radial basis function (RBF) networks, has been developed for quantitative analysis of drugs during the last decade. In this paper, the two components (aspirin and phenacetin) were simultaneously determined in compound aspirin tablets by using near-infrared (NIR) spectroscopy and RBF networks. The total database was randomly divided into a training set (50) and a testing set (17). Different preprocessing methods (standard normal variate (SNV), multiplicative scatter correction (MSC), first-derivative and second-derivative) were applied to two sets of NIR spectra of compound aspirin tablets with different concentrations of two active components and compared each other. After that, the performance of RBF learning algorithm adopted the nearest neighbor clustering algorithm (NNCA) and the criterion for selection used a cross-validation technique. Results show that using RBF networks to quantificationally analyze tablets is reliable, and the best RBF model was obtained by first-derivative spectra. [Copyright &y& Elsevier]

The metabolism of acetyl-labelled phenacetin-C²H3 was investigated in man following a single (150 mg) oral dose. Urine samples were collected at predose, 0-2h and >2-4 h post-dose, and samples from each time-point were then analysed directly using ¹H-nuclear magnetic resonance (NMR) spectroscopy. The phenacetin metabolites acetaminophen (paracetamol) glucuronide, sulphate and the N-acetyl-L-cysteinyl conjugate were identified by this method, and all showed clear evidence of the loss of the original ²H3-acetyl label and its replacement with ¹H3 (futile deacetylation). The observed percentage futile deacetylation by ¹H-NMR spectroscopy was measured as approximately 20% in each metabolite (about 2% of the recovered dose). After sample preparation by solid-phase extraction on a C18 solid-phase extraction (SPE) cartridge, further profiling was performed using high-performance liquid chromatography/mass spectrometry-solid-phase extraction-nuclear magnetic resonance (HPLC/MS-SPE-NMR) confirming futile deacetylation had taken place as indicated by NMR spectroscopy on both the isolated acetaminophen glucuronide and L-cysteinyl-metabolites. Additional analysis by high-performance liquid chromatography-time-of-flight mass spectrometry (HPLC-ToF MS) identified further phenacetin metabolites, and from these data the mean percentage of futile deacetylation was measured as 31%±2% for the acetylated phenacetin metabolites. A number of non-acetylated metabolites were also detected in the sample via HPLC-ToF MS. The results showed that phenacetin underwent a transient formation via a number of toxic intermediates to a much greater extent than had been observed in similar studies on acetaminophen. These results may contribute to the understanding of the analgesic nephropathy reported following chronic phenacetin consumption. [ABSTRACT FROM AUTHOR]

Abstract: Chronic use of phenacetin-containing analgesics has been associated with the development of renal cancer. To establish genotoxicity as a possible cause for the carcinogenic effect of phenacetin, we exposed wild type and DNA repair deficient Xpa −/− and Xpa −/− /Trp53 +/− mice (further referred as Xpa and Xpa/p53 mice, respectively), carrying a reporter lacZ gene, to 0.75% (w/w) phenacetin mixed in feed. Xpa mice completely lack the nucleotide excision repair pathway, and as such they are sensitive to some classes of genotoxic compounds. Phenacetin exposure induced a significant increase of lacZ mutations in the kidney of both Xpa and Xpa/p53 mice. A minor response was found in liver, whereas no lacZ mutation induction was observed in the spleen of these animals. Interestingly, the observed phenacetin-induced mutant frequencies were higher in male than those found in female mice. This gender difference is probably due to a difference in metabolic rate. Phenacetin-induced mutations mainly consisted of point mutations rather than deletions. The mutational spectra in the kidney of treated WT and Xpa mice were quite similar. Taken together, these results demonstrate that the human carcinogen phenacetin acts as a weak genotoxic agent in an in vivo mouse model system. [Copyright &y& Elsevier]