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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.

Indirect oxidation induced by reactive free radicals, such as hydroxyl radical (HO), sulfate radical (SO 4 - ) and carbonate radical (CO 3 - ), plays an important or even crucial role in the degradation of micropollutants. Thus, the coadjutant degradation of phenacetin (PNT) by HO, SO 4 - and CO 3 - , as well as the synergistic effect of O 2 on HO and HO 2 were studied through mechanism, kinetics and toxicity evaluation. The results showed that the degradation of PNT was mainly caused by radical adduct formation (RAF) reaction (69% for Г, the same as below) and H atom transfer (HAT) reaction (31%) of HO. For the two inorganic anionic radicals, SO 4 - initiated PNT degradation by sequential radical addition-elimination (SRAE; 55%), HAT (28%) and single electron transfer (SET; 17%) reactions, while only by HAT reaction for CO 3 - . The total initial reaction rate constants of PNT by three radicals were in the order: SO 4 -  > HO > CO 3 - . The kinetics of PNT degradation simulated by Kintecus program showed that UV/persulfate could degrade target compound more effectively than UV/H 2 O 2 in ultrapure water. In the subsequent reaction of PNT with O 2 , HO and HO 2 , the formation of mono/di/tri-hydroxyl substitutions and unsaturated aldehydes/ketones/alcohols were confirmed. The results of toxicity assessment showed that the acute and chronic toxicity of most products to fish increased and to daphnia decreased, and acute toxicity to green algae decreased while chronic toxicity increased., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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 t 1/2 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 <0.001)., 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., Competing Interests: The authors have no conflicts of interest to declare., (© 2020 Wu et al.)

Recently, a variety of nanoparticles have been widely used as imaging agents or carriers for the diagnosis and therapy of lung cancer. However, their poor imaging effect, high toxicity, pro-inflammatory effect and ineffective treatment are still a great challenge. In this work, we reported a novel kind of BiOI@CuS nanoparticle to achieve safe and effective therapy of lung cancer by co-loading hydrochloric acid doxorubicin (DOX) and aspirin phenacetin and caffeine (APC). The nanoparticles can effectively relieve inflammatory reactions induced by photo-thermal therapy (PTT). In vitro and in vivo assays showed that DOX/APC co-loaded BiOI@CuS exhibited an effective chemo-photothermal comprehensive therapy effect and good CT imaging capability. Consequently, this multifunctional nanosystem provides a versatile and promising platform in the imaging and treatment of lung cancer in further applications.

Purpose: Many bioactive molecules show a type of solution phase behavior, termed promiscuous aggregation, whereby at micromolar concentrations, colloidal drug-rich aggregates are formed in aqueous solution. These aggregates are known to be a major cause of false positives and false negatives in select enzymatic high-throughput screening assays. The goal of this study was to investigate the impact of drug-rich aggregates on in vitro drug screening metabolism assays., Methods: Cilnidipine was selected as an aggregate former and its impact on drug metabolism was evaluated against rCYP2D6, rCYP1A2, rCYP2C9 and human liver microsomes., Results: The cilnidipine aggregates were shown to non-specifically inhibit multiple cytochrome P450 enzymes with an IC 50 comparable with the IC 50 of potent model inhibitors., Conclusions: This newly demonstrated mode of "promiscuous inhibition" is of great importance as it can lead to false positives during drug metabolism evaluations and thus it needs to be considered in the future to better predict in vivo drug-drug interactions.

The purpose of this research was to develop a fiber optic (FO) dissolution method for quantification of multiple actives in combination pharmaceutical tablets. FO dissolution allows direct API quantification in the vessel, obviating the need for error-prone facets of traditional dissolution methods. However, FO dissolution is potentially challenged by overlapping UV spectra, matrix effects, UV-active excipients, API interactions with excipients and media, and undissolved components attenuating the UV signal. These obstacles might render FO dissolution method development more complex than LC-end dissolution. The case study in this manuscript has the added complexity of a triple combination product (Midol), where acetaminophen, caffeine, and pyrilamine maleate exhibit similar release kinetics, share largely overlapping UV spectra and span an order of magnitude difference in concentration. Single-wavelength quantification required unique features for the actives of interest, which were not available for the formulation of interest without preprocessing. The methods employed for the quantification of actives were a partial least squares multivariate calibration and a peak area calibration, both using prepared mixtures as reference data. The selected combination tablet demonstrated collinear API release; therefore, individual quantification required a design of experiments for mixture design. The advantages of FO dissolution will be discussed in the context of the formulation under investigation. Additionally, some general guidelines will be suggested for the development of other FO methods.

Formation of amorphous solid dispersions is an effective way to enhance the bioavailability of drugs. One of the main disadvantages of such systems is their low storage stability. Estimation and prognosis of storage stability of the amorphous solid dispersions are possible through modeling of the kinetics of crystallization by the Arrhenius equation and the resulting parameters, i.e., activation energy and pre-exponential factor. These parameters can be determined using the non-isothermal kinetics methods based on both model-fitting and model-free approaches using the differential scanning calorimetry data. In the present work, the formation of amorphous solid dispersions of the phenacetin model drug with polyvinylpyrrolidone of different molecular masses (3500-1.3 × 10 6  g·mol -1 ) was studied in a wide range of heating and cooling rates. The kinetic parameters of the crystallization process of the active pharmaceutic ingredient in the solid dispersions with increased drug content were determined. The dependence of the kinetic parameters of phenacetin cold crystallization on the molecular weight of the polymer is non-linear. The approaches used in the present work can find applications for the estimation of kinetic stability of amorphous pharmaceutical systems prone to crystallization., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

Leucine382 of cytochrome P450 1A2 (CYP1A2) plays an important role in binding and O -dealkylation of phenacetin, with the L382V mutation increasing substrate oxidation (Huang and Szklarz, 2010, Drug Metab. Dispos . 38:1039⁻1045). This was attributed to altered substrate binding orientation, but no direct experimental evidence had been available. Therefore, in the current studies, we employed nuclear magnetic resonance (NMR) longitudinal (T₁) relaxation measurements to investigate phenacetin binding orientations within the active site of CYP1A2 wild type (WT) and mutants. Paramagnetic relaxation time (T 1P ) for each proton of phenacetin was calculated from the T₁ value obtained from the enzymes in ferric and ferrous-CO state in the presence of phenacetin, and used to model the orientation of phenacetin in the active site. All aromatic protons of phenacetin were nearly equidistant from the heme iron (6.34⁻8.03 Å). In contrast, the distance between the proton of the ⁻OCH₂⁻ group, which is abstracted during phenacetin oxidation, and the heme iron, was much shorter in the L382V (5.93 Å) and L382V/N312L (5.96 Å) mutants compared to the N312L mutant (7.84 Å) and the wild type enzyme (6.55 Å), consistent with modeling results. These studies provide direct evidence for the molecular mechanism underlying increased oxidation of phenacetin upon the L382V mutation.

Inhalation administration is a promising alternative to the invasive drug delivery methods. The particle size required for ideal drug aerosol preparation is between 1 and 3  μ m. The application of microspherical particles of solid dispersions enhances bioavailability of poorly soluble drugs due to the solubilization. In the present work, the spray drying process of the production of microspherical particles of solid dispersions of polyvinylpyrrolidone K29-32 with model hydrophobic drug, phenacetin, was optimized using the results of DSC, PXRD, and viscometry. The diameter of the obtained particles is within 1-3  μ m range. The Gibbs energy of dissolution in water was shown to be negative for the mixture with polymer/phenacetin mass ratio 5 : 1. We have demonstrated that the optimal size distribution for the inhalation administration is obtained for microspherical particles produced using spray caps with 7.0  μ m hole size. The dissolution rates of phenacetin from the produced microspherical particles were faster than that of drug powder. As evidenced by powder X-ray diffraction data, phenacetin stayed in amorphous state for 4 months in microspherical particles of solid dispersions. According to the obtained results, strategic application of the spray drying process could be beneficial for the improvement of the pharmaceutical properties of model drug, phenacetin.

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 (AUC 0-24 ) was significantly (P < 0.0001) increased by 1.4 (L) and 1.3-fold (H), plasma concentration (C max ) was significantly (P < 0.001) increased by 1.6 (L) and 1.4-fold (H). Whereas for TOLBU; clearance rate (CL) was significantly (P < 0.0001) decreased by 2.4 (L) and 2.3-fold (H), C max, was significantly (P < 0.001) decreased by 26.5% (L) and 50.4% (H) and AUC 0-24 was significantly (P < 0.0001) decreased by 59.8% (L) and 57.5% (H). Thus, EL is seen to be interacting with CYP1A2 by inhibiting its metabolic activity. HRLC-MS and GC-MS helped identify the presence of gymnemic acid (GA), triterpenoids and steroids in EL which could be the reason for PK interaction of CYP1A2 and CYP2C9. Also, in silico structure based site of metabolism study showed Fe accessibility and intrinsic activity for GA-IV, GA-VI, GA-VII and GA-X with CYP2C9. PK parameters of AMLO were not significantly affected by pre-treatment of EL. Thereby our findings indicate that co-administration of GS with drugs that are metabolized by CYP2C9 and CYP1A2 could lead to potential HDI., (Copyright © 2017. Published by Elsevier B.V.)

Phenacetin is a pharmaceutical closely related to acetaminophen that has been banned in France for a long time due to its nephritic and carcinogenic adverse effects. It frequently appears in cocaine seizures as a cutting agent. Following both sanitary and intelligence motivations, this molecule was chosen for this study, and stable isotopes seemed to be the most appropriate tool. A total of 228 seized samples were collected over a 6-year period, and 8 standards of known origin were purchased. They were submitted to gas chromatography (GC) or elemental analysis - isotope ratio mass spectrometry (EA-IRMS) measurements, depending on their complexity. Stable isotope ratios of carbon, hydrogen, and nitrogen for a part of the sample set, were acquired. The isotopic values of phenacetin standards acquired from various providers located worldwide are quite spread, which indicates that stable isotopes could be used to discriminate manufacturers. However, the measured values of most of the seized samples are concentrated in a narrow range, tending to demonstrate that phenacetin is smuggled from a single source or similar ones. Consequently, stable isotopes could only be used to exclude that several samples come from a common source. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Phenacetin is a common analgesic, anti-arthritic and anti-rheumatic drug. This study dealt with the degradation of phenacetin in alkaline media using a three-dimensional reactor with particle electrodes. Particular attention was paid to the degradation pathway and the reaction mechanism in the system. Liquid chromatography coupled with time-of-flight mass spectrometry was used to identify the intermediates. The phenacetin was observed to be firstly cut off the branch chains main by direct oxidation, and then the intermediates further degraded to ring opening products by hydroxyl radical resulting from indirect oxidation and finally mineralized to CO2, H2O. A possible removal mechanism was proposed that direct and indirect oxidation together did effect on the pollutants with oxygen., (Copyright © 2015. Published by Elsevier Ltd.)

We present a general framework to predict the excess solubility of small molecular solids (such as pharmaceutical solids) in binary solvents via molecular simulation free energy calculations at infinite dilution with conventional molecular models. The present study used molecular dynamics with the General AMBER Force Field to predict the excess solubility of acetanilide, acetaminophen, phenacetin, benzocaine, and caffeine in binary water/ethanol solvents. The simulations are able to predict the existence of solubility enhancement and the results are in good agreement with available experimental data. The accuracy of the predictions in addition to the generality of the method suggests that molecular simulations may be a valuable design tool for solvent selection in drug development processes.

The kinetics of the dissociation is fundamental to the formation and the in vivo performance of cyclodextrin supramolecules. The individual determination of the apparent dissociation rate constant (kd,app) using high performance affinity chromatography (HPAC) is a tedious process requiring numerous separate studies and massive data fitting. In this study, the multianalyte approach was employed to simultaneously measure the kd,app values of three drugs through one injection based on the investigation of the dependence of drug-cyclodextrin interaction kinetics on the mobile phase composition. As a result, the kd,app values increased when decreasing the ion strength, increasing the ionization of drugs and adding extra organic solvents. The values of kd,app for acetaminophen, phenacetin and S-flurbiprofen estimated by the multianalyte approach were 8.54±1.81, 5.36±0.94 and 0.17±0.02s(-1), respectively, which were in good agreement with those determined separately (8.31±0.58, 5.01±0.42 and 0.15±0.01s(-1)). For both of the single and multiple flow rate peak profiling methods, the results of the multianalyte approach were statistically equivalent with that of the single compound analysis for all of the three drugs (p>0.05). The multianalyte approach can be employed for the efficient evaluation of the drug-cyclodextrin kinetics with less variance caused by cyclodextrin column bleeding., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Phenacetin is known to be metabolized to N-AcetylParaBenzoQuinoneImine (NAPQI), which is a common metabolite of paracetamol (also called acetaminophen or APAP). The electrochemical conversion of APAP to NAPQI was shown in 1989 by Getek and co-workers, thus demonstrating the capacity of electrochemistry to mimic the formation of NAPQI from APAP as well as from phenacetin. This study focuses on a preparative electrochemical electrolysis associated with quantitative (1)H NMR. On one hand, this method is able to synthesize reactive metabolites in sufficient concentrations and amounts for NMR analysis. On the other hand, NMR allows the simultaneous detection and quantification of all chemical species, in contrast to mass spectrometry. The combination of electrochemistry with quantitative NMR is thus presented as a relevant method for elucidating the degradation of reactive metabolites and may be considered a valuable complementary tool to EC-MS., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In situ NMR spectroelectrochemistry is presented in this study as a useful hybrid technique for the chemical structure elucidation of unstable intermediate species. An experimental setting was designed to follow the reaction in real time during the experimental electrochemical process. The analysis of (1)H NMR spectra recorded in situ permitted us (1) to elucidate the reaction pathway of the electrochemical oxidation of phenacetin and (2) to reveal the quinone imine as a reactive intermediate species without using any trapping reaction. Phenacetin has been considered as hepatotoxic at high therapeutic amounts, which is why it was chosen as a model to prove the applicability of the analytical method. The use of 1D and 2D NMR experiments led to the elucidation of the major species produced from the oxidation process. We demonstrated that in situ NMR spectroelectrochemistry constitutes a fast way for monitoring unstable quinone imines and elucidating their chemical structures.

Most pharmaceutical drug substances and excipients in formulations exist in a crystalline or amorphous form, and an understanding of their state during manufacture and storage is critically important, particularly in formulated products. Carbon 13 solid-state nuclear magnetic resonance (NMR) spectroscopy is useful for studying the chemical and physical state of pharmaceutical solids in a formulated product. We developed two new selective signal excitation methods in (13) C solid-state NMR to extract the spectrum of a target component from such a mixture. These methods were based on equalization of the proton relaxation time in a single domain via rapid intraproton spin diffusion and the difference in proton spin-lattice relaxation time in the rotating frame ((1) H T1rho) of individual components in the mixture. Introduction of simple pulse sequences to one-dimensional experiments reduced data acquisition time and increased flexibility. We then demonstrated these methods in a commercially available drug and in a mixture of two saccharides, in which the (13) C signals of the target components were selectively excited, and showed them to be applicable to the quantitative analysis of individual components in solid mixtures, such as formulated products, polymorphic mixtures, or mixtures of crystalline and amorphous phases., (Copyright © 2012 Wiley Periodicals, Inc.)

Human cytochromes P450 1A1 and 1A2 play important roles in drug metabolism and chemical carcinogenesis. Although these two enzymes share high sequence identity, they display different substrate specificities and inhibitor susceptibilities. In the present studies, we investigated the structural basis for these differences with phenacetin as a probe using a number of complementary approaches, such as enzyme kinetics, stoichiometric assays, NMR, and molecular modeling. Kinetic and stoichiometric analyses revealed that substrate specificity (k(cat)/K(m)) of CYP1A2 was approximately 18-fold greater than that of CYP1A1, as expected. Moreover, despite higher H₂O₂ production, the coupling efficiency of reducing equivalents to acetaminophen formation in CYP1A2 was tighter than that in CYP1A1. CYP1A1, in contrast to CYP1A2, displayed much higher uncoupling, producing more water. The subsequent NMR longitudinal (T₁) relaxation studies with the substrate phenacetin and its product acetaminophen showed that both compounds displayed similar binding orientations within the active site of CYP1A1 and CYP1A2. However, the distance between the OCH₂ protons of the ethoxy group (site of phenacetin O-deethylation) and the heme iron was 1.5 Å shorter in CYP1A2 than in CYP1A1. The NMR findings are thus consistent with our kinetic and stoichiometric results, providing a likely molecular basis for more efficient metabolism of phenacetin by CYP1A2.

In conventional metabolism and pharmacokinetic studies, radioactive isotopes are used to identify and quantify the breakdown products of xenobiotics. However, the stable isotope (13) C provides a cheaper and less hazardous alternative. Metabolites of (13) C-enriched xenobiotics can be detected, quantified and identified by (13) C-filtered NMR spectroscopy. However, one obstacle to using (13) C is its 1.1% natural abundance that produces a background signal in (13) C-filtered NMR spectra of crude biological extracts. The signal makes it difficult to distinguish between (13) C-enriched xenobiotics resonances from endogenous metabolites unrelated to the xenobiotic. This study proposes that the (13) C background signal can be distinguished from resonances of (13) C-enriched xenobiotics by the absence of a (12) C component in the xenobiotic. This is detected by combined analysis of (13) C-filtered and -edited NMR spectra. The theory underlying the approach is described and the method is demonstrated by the detection of sub-microgram amounts of (13) C-enriched phenacetin in crude extracts of hepatocyte microsomes., (Copyright © 2012 John Wiley & Sons, Ltd.)

This study centers on the use of inelastic neutron scattering as an alternative tool for physical characterization of solid pharmaceutical drugs. On the basis of such approach, relaxation processes in the pharmaceutical compound phenacetin (p-ethoxyacetanilide, C(10)H(13)NO(2)) were evidenced on heating between 2 and 300 K. By evaluating the mean-square displacement obtained from the elastic fixed window approach, using the neutron backscattering technique, a crossover of the molecular fluctuations between harmonic and nonharmonic dynamical regimes around 75 K was observed. From the temperature dependence of the quasi-elastic line-width, summed over the total Q range explored by the time-of-flight technique, it was possible to attribute the onset of this anharmonicity to methyl group rotations. Finally, using density functional theory-based methods, we were able to calculate the lattice vibrations in the harmonic approximation. The overall spectral profile of the calculated partial contributions to the generalized density of states compares satisfactorily to the experimental spectra in the region of the lattice modes where the intermolecular interactions are expected to play an important role. This study contributes to understanding the relationships between intermolecular hydrogen bonds, intramolecular dynamics, and conformational flexibility in pharmaceuticals on a molecular level, which can help in evaluating phase stability with respect to temperature variations on processing or on storage, and is related to control of polymorphism and pseudopolymorphism.

The empty-level structures and formation of negative ion states via resonance attachment of low-energy (0-15 eV) electrons into vacant molecular orbitals in a series of non-steroidal anti-inflammatory drugs (NSAIDs), namely aspirin, paracetamol, phenacetin, and ibuprofen, were investigated in vacuo by electron transmission and dissociative electron attachment (DEA) spectroscopies, with the aim to model the behavior of these antipyretic agents under reductive conditions in vivo. The experimental findings are interpreted with the support of density functional theory calculations. The negative and neutral fragments formed by DEA in the gas phase display similarities with the main metabolites of these commonly used NSAIDs generated in vivo by the action of cytochrome P450 enzymes, as well as with several known active agents. It is concluded that xenobiotic molecules which possess pronounced electron-accepting properties could in principle follow metabolic pathways which parallel the gas-phase dissociative decay channels observed in the DEA spectra at incident electron energies below 1 eV. Unwanted side effects as, e.g., hepatoxicity or carcinogenicity produced by the NSAIDs under study in human organism are discussed within the "free radical model" framework, reported earlier to describe the toxic action of the well-known model toxicant carbon tetrachloride.

A simple and robust method using high-performance liquid chromatography with UV detection was developed and validated for the determination of six pyrrole-imidazole (PI) polyamides (HN.49, TGF-β1f, TGF-β1t, HN.50f, HN.50t, and LOX-1) in rat plasma. After the plasma proteins were precipitated with methanol containing phenacetin as an internal standard, the analytes were separated on a Luna C18 (2) (5 μm, 4.6 × 150 mm). Calibration curves were linear over the range of 0.5 to 200 μg/mL for HN.49, 0.25 to 200 μg/mL for TGF-β1f, TGF-β1t, HN.50t, and LOX-1, 1 to 200 μg/mL for HN.50f in rat plasma. The inter- and intraday precision were below 15%, and the accuracy was within 15% at the quality controls. The validated method was successfully applied to sample analysis for the pharmacokinetic study.

Electrochemistry in combination with mass spectrometry is emerging as a versatile analytical technique in the imitation of oxidative drug metabolism during the early stages of drug discovery and development. Here, we present electrochemical O-dealkylation of phenacetin to acetaminophen by square-wave potential pulses consisting of consecutive sub-second oxidation and reduction steps. This O-dealkylation could not be achieved by oxidation at constant potential or longer potential pulses because of the fast hydrolysis of the reactive intermediates. Electrochemical conversion by square-wave potential pulses can thus widen the scope of electrochemical synthesis of metabolites and imitation of in vivo drug metabolism.

The bromination of five selected pharmaceuticals (metoprolol, naproxen, amoxicillin, phenacetin, and hydrochlorothiazide) was studied with these compounds individually dissolved in ultra-pure water. The apparent rate constants for the bromination reaction were determined as a function of the pH, obtaining the sequence amoxicillin>naproxen>>hydrochlorothiazide≈phenacetin≈metoprolol. A kinetic mechanism specifying the dissociation reactions and the species formed for each compound according to its pK(a) value and the pH allowed the intrinsic rate constants to be determined for each elementary reaction. There was fairly good agreement between the experimental and calculated values of the apparent rate constants, confirming the goodness of the proposed reaction mechanism. In a second stage, the bromination of the selected pharmaceuticals simultaneously dissolved in three water matrices (a groundwater, a surface water from a public reservoir, and a secondary effluent from a WWTP) was investigated. The pharmaceutical elimination trend agreed with the previously determined rate constants. The influence of the main operating conditions (pH, initial bromine dose, and characteristics of the water matrix) on the degradation of the pharmaceuticals was established. An elimination concentration profile for each pharmaceutical in the water matrices was proposed based on the use of the previously evaluated apparent rate constants, and the theoretical results agreed satisfactorily with experiment. Finally, chlorination experiments performed in the presence of bromide showed that low bromide concentrations slightly accelerate the oxidation of the selected pharmaceuticals during chlorine disinfection., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Poor water solubility leads to low dissolution rate and consequently, it can limit bioavailability. Solid dispersions, where the drug is dispersed into an inert, hydrophilic polymer matrix can enhance drug dissolution. Solid dispersions were prepared using phenacetin and phenylbutazone as model drugs with polyethylene glycol (PEG) 8000 (carrier), by melt fusion method. Phenacetin and phenylbutazone displayed an increase in the dissolution rate when formulated as solid dispersions as compared with their physical mixture and drug alone counterparts. Characterisation of the solid dispersions was performed using differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). DSC studies revealed that drugs were present in the amorphous form within the solid dispersions. FTIR spectra for the solid dispersions of drugs suggested that there was a lack of interaction between PEG 8000 and the drug. However, the physical mixture of phenacetin with PEG 8000 indicated the formation of hydrogen bond between phenacetin and the carrier. Permeability of phenacetin and phenylbutazone was higher for solid dispersions as compared with that of drug alone across Caco-2 cell monolayers. Permeability studies have shown that both phenacetin and phenylbutazone, and their solid dispersions can be categorised as well-absorbed compounds., (Copyright © 2011 Wiley-Liss, Inc.)

A rapid, simple and sensitive HPLC-ESI-MS/MS method was developed for the simultaneous determination of capsaicin and dihydrocapsaicin in rat plasma. Plasma samples containing capsaicin, dihydrocapsaicin and phenacetin (internal standard) were prepared based on a simple protein precipitation by the addition of two volumes of acetonitrile. The analytes and internal standard were separated on a Zorbax SB-C18 column (3.5microm, 2.1mmx100mm) with mobile phase of acetonitrile/water (55:45, v/v) containing 0.1% formic acid (v/v) at a flow rate of 0.2mL/min with an operating temperature of 25 degrees C. Quantification was performed on a triple quadrupole mass spectrometer equipped with electrospray ionization (ESI) source by selected reaction monitoring (SRM) of the transitions at m/z 306-137 for capsaicin, m/z 308-137 for dihydrocapsaicin and m/z 180-110 for the IS. Linear detection responses were obtained for capsaicin and dihydrocapsaicin ranging from 1 to 500ng/mL and the lower limits of quantitation (LLOQs) for the two compounds were 1ng/mL. The intra- and inter-day precisions (R.S.D.%) were within 9.79% for the two analytes, while the deviations of assay accuracies were within +/-10.63%. The average recoveries of the analytes were greater than 89.88%. The analytes were proved to be stable during all sample storage, preparation and analytic procedures. The method was successfully applied to the pharmacokinetic studies of capsaicin and dihydrocapsaicin in rats after subcutaneous administration of capsaicin (natural, containing 65% capsaicin and 35% dihydrocapsaicin)., (Copyright 2010 Elsevier B.V. All rights reserved.)

In this study, the ability of 7 chemically diverse polymers [Eudragit E100 (E100), poly(acrylic acid) (PAA), poly(vinylpyrrolidone) (PVP), poly(vinylpyrrolidone-vinyl acetate) (PVPVA), poly(styrene sulfonic acid) (PSSA), hydroxypropylmethylcellulose (HPMC) and hydroxypropylmethylcellulose acetate succinate (HPMCAS)] to inhibit the crystallization of 8 readily crystallizable model compounds [benzamide (BD), phenacetin (PH), flurbiprofen (FB), flufenamic acid (FFA), chlorpropamide (CP), chlorzoxazone (CZ), bifonazole (BI) and lidocaine (LI)] was investigated. Films of the different drug-polymer combinations were prepared by rapid evaporation from solution, using a spin coating method. A total of 7 different drug/polymer weight ratios [90/10, 75/25, 60/40, 50/50, 40/60, 25/75 and 10/90 (w/w)] were evaluated for each drug-polymer combination. Crystallization behavior of the films was monitored using polarized light microscopy over 7 days of room temperature storage under dry conditions. It was observed that compounds having a higher crystallization tendency for the pure compound tended to be more difficult to stabilize using the polymeric additives; more polymer was required. In addition, the stabilizing ability of the polymers varied considerably for the individual compounds, with the acidic polymers PAA and PSSA showing the most extreme behavior. The acidic polymers were good stabilizers for the drugs with basic and amide functional groups, but extremely poor stabilizers for acidic drugs. A reasonable correlation between crystallization inhibition in spin coated films versus bulk powders (prepared by rotary evaporation) was observed. The small scale screening method is thus a potentially useful technique to evaluate the role of drug-polymer chemistry in the stabilization of amorphous solid dispersions.

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.

Bursera grandifolia and other related species have been used in traditional herbal medicine in Mexico and other Latin American countries for their analgesic, antipyretic and anti-inflammatory properties. From the chloroform extract of leaves of B. grandifolia, a substance was isolated and identified as phenacetin, a well known compound with widely tested analgesic and antipyretic properties. The structural identity of the compound was elucidated on the basis of chemical and spectroscopic evidence and by comparison with an authentic sample.

The solubility of phenacetin, salicylic acid, and probenecid in ethanol-water and ethanol-ethyl acetate mixtures at several temperatures (15-40 degrees 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 delta(1) of both solvent mixtures, at 40% (delta(1) = 21.70 MPa(1/2)) and 30% (delta(1) = 20.91 MPa(1/2)) ethanol in ethyl acetate, respectively. Phenacetin displays two peaks at 60% ethanol in ethyl acetate (23.30 MPa(1/2)) and 90% ethanol in water (delta(1) = 28.64 MPa(1/2)). 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., ((c) 2008 Wiley-Liss, Inc. and the American Pharmacists Association)

Nuclear magnetic resonance (NMR) spectroscopy has traditionally been considered as an indispensable tool in elucidating structures of metabolites. With the advent of Fourier transform (FT) spectrometers, along with improvements in software and hardware (such as high-field magnets, cryoprobes, versatile pulse sequences, and solvent suppression techniques), NMR is increasingly being considered as a critical quantitative tool, despite its lower sensitivity as compared to mass spectrometry. A specific quantitative application of NMR is in determining the concentrations of biologically isolated metabolites, which could potentially be used as reference standards for further quantitative work by liquid chromatography/mass spectrometry. With the recent demands from regulatory agencies on quantitative information on metabolites, it is proposed that NMR will play a significant role in strategies aimed at addressing metabolite coverage in toxicological species. Traditionally, biologically isolated metabolites have not been considered as a way of generating "reference standards" for further quantitative work. However, because of the recent FDA guidance on safety testing of metabolites, one has to consider means of authenticating and quantitating biologically or nonbiologically generated metabolites. 1H NMR is being proposed as the method of choice, as it is able to be used as both a qualitative and a quantitative tool, hence allowing structure determination, purity check, and quantitative measurement of the isolated metabolite. In this publication, the application of NMR as a powerful and robust analytical technique in determining the concentrations of in vitro or in vivo isolated metabolites is discussed. Furthermore, to demonstrate the reliability and accuracy of metabolite concentrations determined by NMR, validation and cross-validation with gravimetric and mass spectrometric methods were conducted.

Cytochrome P450s (P450s) metabolize a large number of diverse substrates with specific regio- and stereospecificity. A number of compounds, including nicotine, cotinine, and aflatoxin B(1), are metabolites of the 94% identical CYP2A13 and CYP2A6 enzymes but at different rates. Phenacetin and 4-aminobiphenyl were identified as substrates of human cytochromes P450 1A2 and 2A13 but not of CYP2A6. The purpose of this study was to identify active site amino acids that are responsible for CYP2A substrate specificity using phenacetin as a structural probe. Ten amino acid residues that differ in the CYP2A13 and CYP2A6 active sites were exchanged between the two enzymes. Phenacetin binding revealed that the six substitution, CYP2A13 S208I, A213S, F300I, A301G, M365V, and G369S decreased phenacetin affinity. Although incorporation of individual CYP2A13 residues into CYP2A6 had little effect on this enzyme's very low levels of phenacetin metabolism, the combination of double, triple, and quadruple substitutions at positions 208, 300, 301, and 369 increasingly endowed CYP2A6 with the ability to metabolize phenacetin. Enzyme kinetics revealed that the CYP2A6 I208S/I300F/G301A/S369G mutant protein O-deethylated phenacetin with a K(m) of 10.3 muM and a k(cat) of 2.9 min(-1), which compare very favorably with those of CYP2A13 (K(m) of 10.7 muM and k(cat) of 3.8 min(-1)). A 2.15 A crystal structure of the mutant CYP2A6 I208S/I300F/G301A/S369G protein with phenacetin in the active site provided a structural rationale for the differences in phenacetin metabolism between CYP2A6 and CYP2A13.

True density is a fundamental and important property of active pharmaceutical ingredients (APIs). Using prediction methods to estimate the API true density can be very beneficial in pharmaceutical research and development, especially when experimental measurements cannot be made due to lack of material or sample handling restrictions. In this paper, two empirical prediction methods developed by Girolami and Immirzi and Perini were used to estimate the true density of APIs, and the estimation results were compared with experimentally measured values by helium pycnometry. The Girolami method is simple and can be used for both liquids and solids. For the tested APIs, the Girolami method had a maximum error of -12.7% and an average percent error of -3.0% with a 95% CI of (-3.8, -2.3%). The Immirzi and Perini method is more involved and is mainly used for solid crystals. In general, it gives better predictions than the Girolami method. For the tested APIs, the Immirzi and Perini method had a maximum error of 9.6% and an average percent error of 0.9% with a 95% CI of (0.3, 1.6%).

The aim of this study was to systematically evaluate the pelletization process parameters of kappa-carrageenan-containing formulations. The study dealt with the effect of 4 process parameters--screw speed, number of die holes, friction plate speed, and spheronizer temperature--on the pellet properties of shape, size, size distribution, tensile strength, and drug release. These parameters were varied systematically in a 2(4) full factorial design. In addition, 4 drugs--phenacetin, chloramphenicol, dimenhydrinate, and lidocaine hydrochloride--were investigated under constant process conditions. The most spherical pellets were achieved in a high yield by using a large number of die holes and a high spheronizer speed. There was no relevant influence of the investigated process parameters on the size distribution, mechanical stability, and drug release. The poorly soluble drugs, phenacetin and chloramphenicol, resulted in pellets with adequate shape, size, and tensile strength and a fast drug release. The salts of dimenhydrinate and lidocaine affected pellet shape, mechanical stability, and the drug release properties using an aqueous solution of pH 3 as a granulation liquid. In the case of dimenhydrinate, this was attributed to the ionic interactions with kappa-carrageenan, resulting in a stable matrix during dissolution that did not disintegrate. The effect of lidocaine is comparable to the effect of sodium ions, which suppress the gelling of carrageenan, resulting in pellets with fast disintegration and drug release characteristics. The pellet properties are affected by the process parameters and the active pharmaceutical ingredient used.

Purpose: We compared the compression properties and characteristics of tablets obtained using the OSDRC system (method (OSDRC)) and conventional compression methods including multi-layer compression with a pharmaceutical powder., Materials and Methods: We prepared tablets using four methods of compression. The force profiles, ejection force, stress relaxation, pressure transmission ratio, and internal intensity of the tablets were measured as compression properties., Results: Method (OSDRC) gave the highest value for the crushing strength of the tablets. Although the compression properties were similar regardless of the method of compression, the internal intensity of tablets compressed by method (OSDRC) was significantly larger than that of the tablets produced by the other methods., Conclusions: In terms of crushing strength, the tablets compressed by method (OSDRC) were superior of those obtained by the conventional compression method. Therefore, it is possible to increase the crushing strength of tablets without changing the pharmaceutical formulation.

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: DeltaGsub298=60.0 kJ/mol; DeltaHsub298=117.9+/-0.7 kJ/mol; DeltaSsub298=190+/-2 J/mol.K; acetanilide: DeltaGsub298=40.5 kJ/mol; DeltaHsub298=99.8+/-0.8 kJ/mol; DeltaSsub298=197+/-2 J/mol.K; phenacetin: DeltaGsub298=52.3 kJ/mol; DeltaHsub298=121.8+/-0.7 kJ/mol; DeltaSsub298=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%)

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 (delta1) 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 delta1=22.59, 21.70 and 20.91 MPa1/2, respectively. Phenacetin displays two solubility maxima, at delta1=25.71 (ethanol-water) and at delta1=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 (delta1=23 MPa1/2). A modification of the extended Hildebrand method is applicable for curves with a single maximum whereas a model including the Hildebrand solubility parameter delta1 and the acidic partial solubility parameter delta1a 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 delta1 of the solvent mixtures. The drugs with solubility parameters below 24 MPa1/2 display a single peak in ethanol-ethyl acetate. The drugs with delta2 values above 25 MPa1/2 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 delta1 values) as the solubility parameter of the drug delta2 increases.

The authors examined the possible peroxyl radical scavanger effect of some non-steroidal antiinflammatory drugs and isomeric dihidroxy benzoic acids by means of the test based on 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-initiated degradation of crocin. The method is based on spectrophotometric determination of crocin, of which rate of AAPH-initiated degradation can be decreased in the presence of antioxidants with peroxyl radical scavanger activity. The authors studied the crocin degradation-inhibitory effect of phenacetin, paracetamol (acetaminophen), indomethacin, ibuprofen, diclofenac sodium, salicylic acid, salicylamide as well as the 2,3-, 2,4-, 2,5- and 3,4-dihydroxy substituted benzoic acids in 200 microM concentration. In the 60 minute studies the 2,3-, 2,5- and 3,4-dihydroxy benzoic acids proved to show the most effective degradation inhibitory effect. The results draw attention to the different antioxidant activity of salicylic acid and its hydroxylated metabolites (2,3- and 2,5-dihydroxy benzoic acids) formed under physiological conditions.

The charge-transfer (CT) interaction of the electron donor phenacetin (phen) and the pi-electron acceptor tetracyanoethylene (TCNE) has been studied in CH(2)Cl(2). The results obtained indicate the formation of the novel CT-complex with the general formula [(phen)(TCNE)(12)]. The 1:12 stoichiometry of the reaction was based on photometric titration, elemental analysis, infrared, thermal and cyclic voltametry measurements of the formed CT-complex.

The molar (K(C)(o/w)) and rational (K(X)(o/w)) partition coefficients in the octanol/buffer, i-propyl myristate/buffer, chloroform/buffer, and cyclohexane/buffer systems were determined for acetanilide and phenacetin at 25.0, 30.0, 35.0, and 40.0 degrees C. In all cases except for cyclohexane, the K(C)(o/w) and K(X)(o/w) values were greater than unity. This demonstrates that these two drugs have predominantly lipophilic behavior. Gibbs and van't Hoff thermodynamic analyses have revealed that the transfer of these drugs from water to organic solvents is spontaneous and that it is mainly driven enthalpically for i-propyl myristate and chloroform, and entropy-driven for octanol and cyclohexane.

High-performance liquid chromatography (HPLC) combined with inductively coupled plasma mass spectrometry (ICPMS) has been studied as a means for the detection of carbon to provide a 'universal' method for detecting organic compounds in chromatographic eluents. Carbon is particularly difficult to ionise and the amount of carbon present in normal chromatographic systems leads to high backgrounds, making detection a challenge. Novel separation approaches were therefore employed, using either entirely aqueous eluents (at temperatures of 60 and 160 degrees C, dependent on the column used) to eliminate the organic modifier completely, or isotopically enriched solvents. For the aqueous eluents, detection limits for sulphanilamide were found to be 2.26 microg, corresponding to 1.13 micromol (0.47 micromol of carbon), injected on a conventional 4.6 mm i.d. column. The use of a narrow bore column with highly isotopically enriched 12C-methanol (99.95 atom%) as organic modifier for the mobile phase enabled the detection of 86 micromol for 13C-triple-labelled caffeine and 79 micromol for 13C-double-labelled phenacetin. The sensitive detection of 12C-compounds with 13C-enriched methanol as organic modifier proved impractical due to a lower level of isotopic enrichment (99 atom%) of this solvent, with the residual 12C-methanol resulting in significant interference., (Copyright 2004 John Wiley & Sons, Ltd.)

The present study deals with controlled drug delivery from hydrocolloid tablets by polymer particle erosion. The influence of excipients and formulation factors on the dissolution behaviour of the methyl hydroxyethyl cellulose (MHEC)-tablets is investigated. Linear drug release with low susceptibility to hydrodynamic stress is obtained. The use of drugs with higher solubility leads to a slight acceleration of the release due to the contribution of diffusion to the release process. Higher drug loading and consequently lower polymer content expedites dissolution as well as changes in the tablets' geometry resulting in enlarged release surfaces. Furthermore, alterations of the composition of the dissolution medium affect drug release. However, neither viscosity grade nor the particle size of the polymer or compaction pressure has a marked impact on the dissolution. Investigations to clarify the mechanism of polymer particle erosion include erosion studies and the comparison of different batches of MHEC, of products from different manufacturers and of fibrous trial products. There is evidence that the insoluble fibres within the water soluble MHEC are responsible for the occurrence of polymer particle erosion by disturbing swelling and formation of a thick coherent gel layer and thus, causing erosion of the hydrocolloid tablet with synchronous drug release.

Human cytochrome P450 (P450) 1A2 is involved in the oxidation of many important drugs and carcinogens. The prototype substrate phenacetin is oxidized to an acetol as well as the O-dealkylation product [Yun, C.-H., Miller, G. P., and Guengerich, F. P. (2000) Biochemistry 39, 11319-11329]. In an effort to improve rates of catalysis of P450 1A2 enzymes, we considered a set of p-alkoxyacylanilide analogues of phenacetin and found that variations in the O-alkyl and N-acyl substituents altered the rates of the two oxidation reactions and the ratio of acetol/phenol products. Moving one methylene group of phenacetin from the O-alkyl group to the N-acyl moiety increased rates of both oxidations approximately 5-fold and improved the coupling efficiency (oxidation products formed/NADPH consumed) from 6% to 38%. Noncompetitive kinetic deuterium isotope effects of 2-3 were measured for all O-dealkylation reactions examined with wild-type P450 1A2 and the E225I mutant, which has 6-fold higher activity. A trend of decreasing kinetic deuterium isotope effect for E225I > wild-type > mutant D320A was observed for O-demethylation of p-methoxyacetanilide, which follows the trend for k(cat). The set of O-dealkylation and acetol formation results for wild-type P450 1A2 and the E225I mutant with several of the protiated and deuterated substrates were fit to a model developed for the basic catalytic cycle and a set of microscopic rate constants in which the only variable was the rate of product formation (substrate oxygenation, including hydrogen abstraction). In this model, k(cat) is considerably less than any of the microscopic rate constants and is affected by several individual rate constants, including the rate of formation of the oxygenating species, the rate of substrate oxidation by the oxygenating species, and the rates of generation of reduced oxygen species (H(2)O(2), H(2)O). This analysis of the effects of the individual rate constants provides a framework for consideration of other P450 reactions and rate-limiting steps.

We previously reported that sodium citrate (Na citrate), which is a high order salt in the Hofmeister's series, greatly suppressed particle agglomeration in fluidized bed coating (Pharm. Res., 16 (1999), 1616-1620). In this paper, we studied the effects of Na citrate concentration on the particle agglomeration in fluidized bed coating and on the structure of coated film on the particles. Spherical granules made of crystalline cellulose (Celphere) containing phenacetin were coated in a fluidized bed with the aqueous coating solution of hydroxypropylmethyl cellulose (HPMC) containing Na citrate at various concentrations. The particle diameter and drug release profile of coated particles, and the physical properties, i.e. tensile strength, elongation percentage at break, porosity and pore size distribution, of the HPMC cast film were investigated. The particle agglomeration was suppressed with the increasing Na citrate concentration. It is considered that the increase in the suppression effect was caused by the salting-out effect of the increased Na citrate. In the HPMC cast film system, the tensile strength and elongation percentage decreased and the porosity and cumulative pore volume increased with an increase in Na citrate concentration. It is considered that the increase in the porosity by adding Na citrate resulted from a phase separation due to the salting-out during the film forming process. The drug release rate from coated particles also increased with the increasing Na citrate concentration. It can be concluded that the increase in the release rate was due to the increase in porosity of the HPMC coated film caused by the increased Na citrate concentration.

The competitive inclusion complexations in the ternary phenacetin/competitors/beta-cyclodextrin (beta-CyD) systems were investigated by the solubility method, where m-bromobenzoic acid (m-BBA) and o-toluic acid (o-TA) were used as competitors. The solubility changes of the drug and competitors as a function of beta-CyD concentration in the ternary systems were formulated using their stability constants and intrinsic solubilities. The decrease in solubility of phenacetin by the addition of competitors could be quantitatively simulated by the formulation, when both drug and competitor give A(L) type solubility diagrams. On the other hand, when one of the guests gives a B(S) type solubility diagram, its solubility change was clearly reflected in that of the another guest, i.e., phenacetin gave an A(L) type solubility diagram in the binary phenacetin/beta-CyD system and o-TA gave a B(S) type diagram in the binary o-TA/beta-CyD system, but in the ternary phenacetin/o-TA/beta-CyD system, a new plateau region appeared in the original A(L) type diagram of phenacetin. This was explained by the solubilization theory of Higuchi and Connors. The solubility analysis of the ternary drug/competitor/CyD systems may be particularly useful for determination of the stability constant of a drug whose physicochemical and spectroscopic analyses are difficult, because they can be calculated by monitoring the solubility change of a competitor, without monitoring that of a drug. Furthermore, the present results suggest that attention should be paid to the type of the phase solubility diagram, as well as the magnitude of the stability constant and the solubility of the complex, for a rational formulation design of CyD complexes.

Mutants with altered activities were obtained from random libraries of human cytochrome P450 (P450) 1A2 with the putative substrate recognition sequences (SRS) mutated [Parikh, A., Josephy, P. D., and Guengerich, F. P. (1999) Biochemistry 38, 5283-5289]. Six mutants from SRS 2 (E225I, E225N, F226I, and F226Y) and 4 (D320A and V322A) regions were expressed as oligohistidine-tagged proteins, purified to homogeneity, and used to analyze kinetics of individual steps in the catalytic cycle, to determine which reaction steps have been altered. When the wild-type, E225I, E225N, F226I, F226Y, D320A, and V322A proteins were reconstituted with NADPH-P450 reductase, rates of 7-ethoxyresorufin O-deethylation and phenacetin O-deethylation were in accord with those expected from membrane preparations. Within each assay, the values of k(cat)/K(m) varied by 2-3 orders of magnitude, and in the case of E225I and E225N, these parameters were 7-8-fold higher than for the wild-type enzyme. The coupling efficiency obtained from the rates of product formation and NADPH oxidation was low (<20%) in all enzymes. No correlation was found between activities and several individual steps in the catalytic cycle examined, including substrate binding, reduction kinetics, NADPH oxidation, and H(2)O(2) formation. Quench reactions did not show a burst for either phenacetin O-deethylation or formation of the acetol, a minor product, indicating that rate-determining steps occur prior to product formation. Inter- and intramolecular kinetic deuterium isotope effects for phenacetin O-deethylation were 2-3. In the case of phenacetin acetyl hydroxylation (acetol formation), large isotope effects [(D)k(cat) or (D)(k(cat)/K(m)) > 10] were observed, providing evidence for rate-limiting C-H bond cleavage. We suggest that the very high isotope effect for acetol formation reflects rate-limiting hydrogen atom abstraction; the lower isotope effect for O-deethylation may be a consequence of a 1-electron transfer pathway resulting from the low oxidation potential of the substrate phenacetin. These pre-steady-state, steady-state, and kinetic hydrogen isotope effect studies indicate that the rate-limiting steps are relatively unchanged over an 800-fold range of catalytic activity. We hypothesize that these SRS mutations alter steps leading to the formation of the activated Michaelis complex following the introduction of the first electron.

1. The hydroxamic acids N-hydroxyphenacetin and N-hydroxy-2-acetylaminofluorene were reduced to the corresponding amides, phenacetin and 2-acetylaminofluorene respectively by rabbit blood supplemented with both NAD(P)H and FAD. These reducing activities were found in erythrocytes but not in plasma, and were sensitive to inhibition by carbon monoxide and oxygen. When blood or erythrocytes were boiled, these activities were not abolished. 2. Haemoproteins such as haemoglobin and catalase exhibited the reductase activity in the presence of both NAD(P)H and FAD under anaerobic conditions. The activity was not abolished when the haemoproteins were boiled. 3. Haematin showed a significant reducing activity in the presence of these cofactors. The activity of haematin was also observed with the photochemically reduced form of FAD. 4. The reduction system in blood was composed of NAD(P)H, FAD and haemoglobin. Reduction appears to proceed in two steps, i.e. the reduction of FAD by NADH or NADPH, followed by the non-enzymatic reduction of the hydroxamic acids to the amides by reduced FAD, catalyzed by the haem group of haemoglobin in rabbit erythrocytes.

Solid dispersion composed of the poly(ethylene oxide) (PEO)-Carbopol((R)) (CP) interpolymer complex containing phenacetin (PHE) was prepared by using six grades of CP having various cross-linking degrees. We attempted to control the medicine release from the PEO-CP solid dispersion by varying the CP grade. The powder X-ray diffraction pattern and differential scanning calorimetry curves suggested that PHE existed in the amorphous state, and PEO in the crystalline state disappeared in the solid dispersions. The release profile of PHE varied depending on the CP grade. A small release rate was observed at CP910 and CP971P that are cross-linked at low and middle degrees, respectively. The Fourier transform-infrared (FT-IR) spectra showed that the amount of the PEO-CP complex formed by hydrogen bonding changed depending on the CP grade. With the cross-linked CP, a good correlation was observed between the hydrogen bonding percent and the percent released of the PHE after 60 min (D(60 min)), indicating that PHE release was controlled by the amount of PEO-CP complex formation in the solid dispersion. These results show that it is feasible to control the medicine release from PEO-CP solid dispersion by varying the CP grade.