[Comparison of the early analgesic efficacy of three different drugs after anterior cruciate ligament reconstruction].

OBJECTIVE: The pain-relieving effect and safety of compound aminopyrine phenacetin tablets, tramcontin (tramadol hydrochloride sustained-release tablets) and dolantin in the early stage of autologous tendon reconstruction of the anterior cruciate ligament (ACL) of the knee joint were compared. METHODS: Retrospective analysis of postoperative pain and drug analgesia in 45 patients performed by the same group from November 2018 to February 2019. The random area group design was divided into two groups according to whether ACL rupture was combined with meniscal injury, group A was 24 patients with ACL reconstruction of knee joint and group B was 21 patients with ACL fracture combined with meniscus injury. The two groups were divided into three subgroups respectively according to the actual treatment of postoperative analgesic drugs received by the patients, including 4 cases of compound aminopyrine phenacetin tablets, 11 cases of oral tramcontin, 9 cases of intramuscular dolantin combined with phenergan in group A; 3 cases of compound aminopyrine phenacetin tablets, 10 cases of oral tramcontin, and 8 cases of intramuscular dolantin combined with phenergan in group B. When the early postoperative patients complain about pain and actively ask for analgesia. When the patients complained about pain after the operation and actively asked for analgesia, they were randomly given painkillers, tramcontin or dolantin combined with phenergan to relieve pain. Pain visual analogue scale (VAS) was used to evaluate pain relief and observe the occurrence of adverse reactions. RESULTS: There were no significant dif-ferences in gender, age, body mass index, and time of hospital stay between the two groups of patients (P > 0.05). In the patients who used tramcontin and dolantin combined with phenergan to relieve pain judging by VAS score before and 1 h after taking the drug, it was found that the pain situation of the patient was significantly relieved, and the difference before and after taking the drug had statistical significance (P < 0.05). Pairwise comparisons of the three drugs applied in the two groups showed significantly greater pain relief in the dolantin combined with phenergan group than in the remaining two drugs. There was no significant difference (P > 0.05). Dolantin was prone to nausea and vomiting, but the application of phenergan was also used to reduce side effects. In terms of adverse reactions, only 1 case of nausea occurred in the tramcontin group for simple ACL reconstruction, and none of the patients in the other groups showed serious complications and allergic reactions. CONCLUSION: Whether in cruciate ligament reconstruction alone or combined with meniscus molding or suture, compound aminopyrine phenacetin tablets, tramcontin, dolantin combined with phenergan can effectively relieve pain. Among the three drugs, dolantin caused the largest pain relief. At the same time, the combination of phenergan effectively reduced the adverse reactions, such as vomiting and nausea, and increased the drug safety.

Newly Identified Tree Shrew Cytochrome P450 2A13 is Expressed in Liver and Lung and Encodes a Functional Drug-Metabolizing Enzyme Similar to Dog Cytochrome P450 2A13 and Pig Cytochrome P450 2A19.

The tree shrew, a non-rodent primate-like species, is used in various fields of biomedical research, including hepatitis virus infection, myopia, depression, and toxicology. Recent genome analysis found that the numbers of cytochrome P450 (P450 or CYP) genes are similar in tree shrews and humans and their sequence identities are high. Although the P450s are a family of important drug-metabolizing enzymes, they have not yet been fully investigated in tree shrews. In the current study, tree shrew CYP2A13 cDNA was isolated from liver, and its characteristics were compared with those of pig, dog, and human CYP2As. Tree shrew CYP2A13 amino acid sequences were highly identical (87-92%) to the human CYP2As and contained sequence motifs characteristic of P450s. Phylogenetic analysis revealed that tree shrew CYP2A13 was more closely related to human CYP2As than to rat CYP2As, similar to dog and pig CYP2As. Among the tissue types analyzed, tree shrew CYP2A13 mRNA was preferentially expressed in liver and lung, similar to dog CYP2A13 mRNA, whereas dog CYP2A25 and pig CYP2A19 mRNAs were predominantly expressed in liver. Tree shrew liver microsomes and tree shrew CYP2A13 proteins heterologously expressed in Escherichia coli catalyzed coumarin 7-hydroxylation and phenacetin O-deethylation, just as human, dog, and pig CYP2A proteins and liver microsomes do. These results demonstrate that tree shrew CYP2A13 is expressed in liver and lung and encodes a functional drug-metabolizing enzyme. SIGNIFICANCE STATEMENT: Novel tree shrew cytochrome P450 2A13 (CYP2A13) was identified and characterized in comparison with human, dog, and pig CYP2As. Tree shrew CYP2A13 isolated from liver had high sequence identities and close phylogenetic relationships to its human homologs and was abundantly expressed in liver and lung at the mRNA level. Tree shrew CYP2A13 metabolized coumarin and phenacetin, human selective CYP2A6 and CYP2A13 substrates, respectively, similar to dog and pig CYP2As, and is a functional drug-metabolizing enzyme likely responsible for drug clearances.

Selective inhibitory effects of suberosin on CYP1A2 in human liver microsomes.

Suberosin is a natural phytoconstituent isolated from Citropsis articulata, especially employed for its anticoagulant properties. Although metabolic studies assessing suberosin have been conducted, it is possible interactions with drugs and food have not yet been investigated. In the present study, we analyzed the selective inhibitory effects of suberosin on cytochrome P450 (CYP) enzymes using a cocktail probe assay. Various concentrations of suberosin (0-50 µM) were incubated with isoform-specific CYP probes in human liver microsomes (HLMs). We found that suberosin significantly inhibited CYP1A2-catalyzed phenacetin O-deethylation, exhibiting IC50 values of 9.39 ± 2.05 and 3.07 ± 0.45 µM with and without preincubation in the presence of ß-NADPH, respectively. Moreover, suberosin showed concentration-dependent, but not time-dependent, CYP1A2 inhibition in HLMs, indicating that suberosin acts as a substrate and reversible CYP1A2 inhibitor. Using a Lineweaver-Burk plot, we found that suberosin competitively inhibited CYP1A2-catalyzed phenacetin O-deethylation. Furthermore, suberosin showed similar inhibitory effects on recombinant human CYP1A1 and 1A2. In conclusion, suberosin may elicit herb-drug interactions by selectively inhibiting CYP1A2 during the concurrent administration of drugs that act as CYP1A2 substrates.

Co3O4 anchored on biochar derived from chitosan (Co3O4@BCC) as a catalyst to efficiently activate peroxymonosulfate (PMS) for degradation of phenacetin.

Chitosan, as a bio-friendly and abundant biochar precursor, was employed to prepare cobalt-based catalyst (Co3O4@BCC) by calcination for activating peroxymonosulfate (PMS) to degrade phenacetin (PNT). Various characterization technologies and experimental designs were performed to investigate the physicochemical properties and catalytic performance of Co3O4@BCC. Approximately 99.0% of phenacetin (10 mg/L) was degraded in the system of Co3O4@BCC (0.05 g/L)/PMS (1.0 mM) within 15 min and the rate constant was 6 times higher than that in the system of Co3O4 (0.05 g/L)/PMS (1.0 mM). The results demonstrated that BCC as a carrier not only dispersed Co3O4 nanoparticles and improved the stability of catalyst, but also provided abundant electron-rich groups to facilitate the activation of PMS and the production of reactive oxygen species (ROS). Co3O4@BCC composite also exhibited good universality and reusability. More than 90% of BPA, SIZ and CAP was degraded by Co3O4@BCC activated PMS within 15 min at pH 7. The degradation rate of PNT was recovered from 90% to 98.0% via the regeneration of the used catalyst after the third run (calcination at 400 °C for 5 min). SO4•-, •OH and 1O2 were identified to be responsible for PNT degradation. Furthermore, the activation mechanism of PMS and the possible pathways of PNT degradation were reasonably speculated according to the results of electron paramagnetic resonance (EPR), X-ray photoelectron spectroscopy (XPS), quenching experiments and HPLC-TOF-MS2. This study explored the application of chitosan as a recycled material and provides a feasible strategy for designing and fabricating environmentally friendly and efficient catalysts for PMS activation to degrade organic pollutants.

Analytical Method Development of Benzisothiazolinone, a Biocide, Using LC-MS/MS and a Pharmacokinetic Application in Rat Biological Matrices.

Benzisothiazolinone (BIT), a biocide widely used as a preservative in household cleaning and personal care products, is cytotoxic to lung cells and a known skin allergen in humans, which highlights the importance of assessing its toxicity and pharmacokinetics. In this study, a simple, sensitive, and accurate LC−MS/MS method for the quantification of BIT in rat plasma, urine, or tissue homogenates (50 µL) using phenacetin as an internal standard was developed and validated. Samples were extracted with ethyl acetate and separated using a Kinetex phenyl−hexyl column (100 × 2.1 mm, 2.6 µm) with isocratic 0.1% formic acid in methanol and distilled water over a run time of 6 min. Positive electrospray ionization with multiple reaction monitoring transitions of m/z 152.2 > 134.1 for BIT and 180.2 > 110.1 for phenacetin was used for quantification. This assay achieved good linearity in the calibration ranges of 2−2000 ng/mL (plasma and urine) and 10−1000 ng/mL (tissue homogenates), with r ≥ 0.9929. All validation parameters met the acceptance criteria. BIT pharmacokinetics was evaluated via an intravenous and dermal application. This is the first study that evaluated BIT pharmacokinetics in rats, providing insights into the relationship between BIT exposure and toxicity and a basis for future risk assessment studies in humans.

Acetaminophen Has Lipid Composition-Dependent Membrane Interactivity That Could Be Related to Nephrotoxicity but Not to Analgesic Activity and Hepatotoxicity.

OBJECTIVE: Although acetaminophen is one of the most widely used over-the-counter drugs, the mechanisms by which this classical drug exerts analgesic, hepatotoxic, and nephrotoxic effects remain unclear. We hypothesized that acetaminophen might act on cellular membranes of nerves, liver, and kidneys. In order to verify this hypothesis, we studied the interactivity of acetaminophen with biomimetic lipid bilayer membranes by comparing with structurally related phenacetin. METHODS: Liposomal membranes (unilamellar vesicles suspended in the buffer of pH 7.4) were prepared with phospholipids and cholesterol to mimic the membrane lipid composition of neuronal cells, hepatocytes, and nephrocytes. They were subjected to reactions with acetaminophen and phenacetin at clinically relevant concentrations, followed by measuring fluorescence polarization to determine their membrane interactivity to modify membrane fluidity. RESULTS: Acetaminophen and phenacetin interacted with neuro-mimetic and hepato-mimetic membranes to increase membrane fluidity at 10-100 µM. Both drugs were more effective in fluidizing hepato-mimetic membranes than neuro-mimetic membranes. Although the relative membrane-interacting potency was phenacetin >> acetaminophen in neuro-mimetic and hepato-mimetic membranes, such membrane effects conflicted with their relative analgesic and hepatotoxic effects. Acetaminophen and phenacetin strongly interacted with nephro-mimetic membranes to increase membrane fluidity at 2-100 µM and 0.1-100 µM, respectively. Phenacetin interacted significantly with nephro-mimetic membranes at lower concentrations (<2 µM) than acetaminophen, which was consistent with their relative nephrotoxic effects. CONCLUSION: In comparison with phenacetin, lipid composition-dependent membrane interactivity of acetaminophen could be related to nephrotoxicity but not to analgesic activity and hepatotoxicity.

Simultaneous detection of phenacetin and paracetamol using ELISA and a gold nanoparticle-based immunochromatographic test strip.

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

Thermodynamic Characteristics of Phenacetin in Solid State and Saturated Solutions in Several Neat and Binary Solvents.

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.

Drug-Related Carcinogenesis: Risk Factors and Approaches for Its Prevention.

The review summarizes the data on the role of metabolic and repair systems in the mechanisms of therapy-related carcinogenesis and the effect of their polymorphism on the cancer development risk. The carcinogenic activity of different types of drugs, from the anticancer agents to analgesics, antipyretics, immunomodulators, hormones, natural remedies, and non-cancer drugs, is described. Possible approaches for the prevention of drug-related cancer induction at the initiation and promotion stages are discussed.

Theoretical investigation on the contribution of HO, SO4- and CO3- radicals to the degradation of phenacetin in water: Mechanisms, kinetics, and toxicity evaluation.

Indirect oxidation induced by reactive free radicals, such as hydroxyl radical (HO), sulfate radical (SO4-) and carbonate radical (CO3-), plays an important or even crucial role in the degradation of micropollutants. Thus, the coadjutant degradation of phenacetin (PNT) by HO, SO4- and CO3-, as well as the synergistic effect of O2 on HO and HO2 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, SO4- 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 CO3-. The total initial reaction rate constants of PNT by three radicals were in the order: SO4- > HO > CO3-. The kinetics of PNT degradation simulated by Kintecus program showed that UV/persulfate could degrade target compound more effectively than UV/H2O2 in ultrapure water. In the subsequent reaction of PNT with O2, HO and HO2, 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.

Arylacetamide Deacetylase Enzyme: Presence and Interindividual Variability in Human Lungs.

Human arylacetamide deacetylase (AADAC) is a single microsomal serine esterase involved in the hydrolysis of many acetyl-containing drugs. To date, the presence and activity of the AADAC enzyme in human lungs has been scarcely examined. We investigated its gene and protein expression as well as interindividual variations in AADAC activities in a large number of human lungs (n = 25) using phenacetin as a selective substrate. The kinetic parameters K m and V max were determined. Our findings highlighted a high interindividual variability in both AADAC mRNA levels and hydrolysis activities. Furthermore, for the first time we demonstrated the presence of the AADAC protein in various lung samples by means of immunoblot analysis. As a comparison, phenacetin hydrolysis was detected in pooled human liver microsomes. Lung activities were much lower than those found in the liver. However, similar K m values were found, which suggests that this hydrolysis could be due to the same enzyme. Pulmonary phenacetin hydrolysis proved to be positively correlated with AADAC mRNA (*P < 0.05) and protein (*P < 0.05) levels. Moreover, the average values of AADAC activity in smokers was significantly higher than in nonsmoker subjects (*P < 0.05), and this might have an important role in the administration of some drugs. These findings add more information to our knowledge of pulmonary enzymes and could be particularly useful in the design and preclinical development of inhaled drugs. SIGNIFICANCE STATEMENT: This study investigated the presence and activity of the AADAC enzyme in several human lungs. Our results highlight high interindividual variability in both AADAC gene and protein expression as well as in phenacetin hydrolysis activity. These findings add more information to our knowledge of pulmonary enzymes and could be particularly useful in the design and preclinical development of inhaled drugs.

The Effect of Promiscuous Aggregation on in Vitro Drug Metabolism Assays.

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 IC50 comparable with the IC50 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.

Determination of cocaine adulterants in human urine by dispersive liquid-liquid microextraction and high-performance liquid chromatography.

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

Multivariate approaches for the development of quality control in-situ fiber optics dissolution methods for fixed-dose combination tablets.

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.

Increased Phenacetin Oxidation upon the L382V Substitution in Cytochrome P450 1A2 is Associated with Altered Substrate Binding Orientation.

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 (T1P) 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.

Modulation of Rat Hepatic CYP1A and 2C Activity by Honokiol and Magnolol: Differential Effects on Phenacetin and Diclofenac Pharmacokinetics In Vivo.

Honokiol (2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol) and magnolol (4-Allyl-2-(5-allyl-2-hydroxy-phenyl)phenol) are the major active polyphenol constituents of Magnolia officinalis (Magnoliaceae) bark, which has been widely used in traditional Chinese medicine (Houpu Tang) for the treatment of various diseases, including anxiety, stress, gastrointestinal disorders, infection, and asthma. The aim of this study was to investigate the direct effects of honokiol and magnolol on hepatic CYP1A and 2C-mediated metabolism in vitro using rat liver microsomes and in vivo using the Sprague-Dawley rat model. Honokiol and magnolol inhibited in vitro CYP1A activity (probe substrate: phenacetin) more potently than CYP2C activity (probe substrate: diclofenac): The mean IC50 values of honokiol for the metabolism of phenacetin and diclofenac were 8.59 µM and 44.7 µM, while those of magnolol were 19.0 µM and 47.3 µM, respectively. Notably, the systemic exposure (AUC and Cmax) of phenacetin, but not of diclofenac, was markedly enhanced by the concurrent administration of intravenous honokiol or magnolol. The differential effects of the two phytochemicals on phenacetin and diclofenac in vivo pharmacokinetics could at least be partly attributed to their lower IC50 values for the inhibition of phenacetin metabolism than for diclofenac metabolism. In addition, the systemic exposure, CL, and Vss of honokiol and magnolol tended to be similar between the rat groups receiving phenacetin and diclofenac. These findings improve our understanding of CYP-mediated drug interactions with M. officinalis and its active constituents.

Effects of MicroRNA-34a on the Pharmacokinetics of Cytochrome P450 Probe Drugs in Mice.

MicroRNAs (miRNAs or miRs), including miR-34a, have been shown to regulate nuclear receptor, drug-metabolizing enzyme, and transporter gene expression in various cell model systems. However, to what degree miRNAs affect pharmacokinetics (PK) at the systemic level remains unknown. In addition, miR-34a replacement therapy represents a new cancer treatment strategy, although it is unknown whether miR-34a therapeutic agents could elicit any drug-drug interactions. To address this question, we refined a practical single-mouse PK approach and investigated the effects of a bioengineered miR-34a agent on the PK of several cytochrome P450 probe drugs (midazolam, dextromethorphan, phenacetin, diclofenac, and chlorzoxazone) administered as a cocktail. This approach involves manual serial blood microsampling from a single mouse and requires a sensitive liquid chromatography-tandem mass spectrometry assay, which was able to illustrate the sharp changes in midazolam PK by ketoconazole and pregnenolone 16α-carbonitrile as well as phenacetin PK by α-naphthoflavone and 3-methylcholanthrene. Surprisingly, 3-methylcholanthrene also decreased systemic exposure to midazolam, whereas both pregnenolone 16α-carbonitrile and 3-methylcholanthrene largely reduced the exposure to dextromethorphan, diclofenac, and chlorzoxazone. Finally, the biologic miR-34a agent had no significant effects on the PK of cocktail drugs but caused a marginal (45%-48%) increase in systemic exposure to midazolam, phenacetin, and dextromethorphan in mice. In vitro validation of these data suggested that miR-34a slightly attenuated intrinsic clearance of dextromethorphan. These findings from single-mouse PK and corresponding mouse liver microsome models suggest that miR-34a might have minor or no effects on the PK of coadministered cytochrome P450-metabolized drugs.

In vitro Phase I- and Phase II-Drug Metabolism in The Liver of Juvenile and Adult Göttingen Minipigs.

PURPOSE: In view of pediatric drug development, juvenile animal studies are gaining importance. However, data on drug metabolizing capacities of juvenile animals are scarce, especially in non-rodent species. Therefore, we aimed to characterize the in vitro biotransformation of four human CYP450 substrates and one UGT substrate in the livers of developing Göttingen minipigs. METHODS: Liver microsomes from late fetal, Day 1, Day 3, Day 7, Day 28, and adult male and female Göttingen minipigs were incubated with a cocktail of CYP450 substrates, including phenacetin, tolbutamide, dextromethorphan, and midazolam. The latter are probe substrates for human CYP1A2, CYP2C9, CYP2D6, and CYP3A4, respectively. In addition, the UGT multienzyme substrate (from the UGT-GloTM assay), which is glucuronidated by several human UGT1A and UGT2B enzymes, was also incubated with the porcine liver microsomes. RESULTS: For all tested substrates, drug metabolism significantly rose postnatally. At one month of age, 60.5 and 75.4% of adult activities were observed for acetaminophen and dextrorphan formations, respectively, while 35.4 and 43.2% of adult activities were present for 4-OH-tolbutamide and 1'-OH-midazolam formations. Biotransformation of phenacetin was significantly higher in 28-day-old and adult females compared with males. CONCLUSIONS: Maturation of metabolizing capacities occurred postnatally, as described in man.

Preoperative Phenacetin Metabolism Test in the Prediction of Postoperative Liver Dysfunction of Patients with Hepatocellular Carcinoma.

BACKGROUND The risk of postoperative liver dysfunction (PLD) in patients with injured livers, such as in hepatocellular carcinoma (HCC), is still not negligible. Phenacetin metabolism test can reflect hepatic functional reserve in patients with chronic hepatic damage. The aim of this study was to assess the ability of phenacetin metabolism test to predict PLD in patients with HCC receiving partial hepatectomy. MATERIAL AND METHODS Forty-nine patients with HCC undergoing partial hepatectomy between 2014 and 2016 were included at Huashan Hospital, Fudan University. The phenacetin metabolism test was used to assess the hepatic functional reserve. The ratio of total plasma paracetamol to phenacetin was collected in patients at 2 h after oral administration of 1.0 g phenacetin, recorded 5 days prior to surgery and on the fifth postoperative day. Phenacetin metabolism test, Child-Pugh classification, and Model for End-Stage Liver Disease (MELD) score were correlated with PLD. RESULTS Of 49 patients with HCC, 13 patients (26.5%) had PLD. The association between the ratio of total plasma paracetamol to phenacetin and PLD was statistically significant (p=0.0061) and the correlation coefficient was -0.647 (p=0.0082). The phenacetin metabolism test showed a larger area under the receiver operating characteristic (ROC) curve value (0.735) than Child-Pugh's classification (0.472) and MELD score (0.419). Using the calculated cutoff of 0.6, the lower ratio of total plasma paracetamol to phenacetin preoperatively was chosen to specifically identify patients with PLD. The sensitivity and specificity were 0.657 and 0.892, respectively. CONCLUSIONS Phenacetin metabolism test could be preoperatively used in predicting PLD in HCC patients receiving partial hepatectomy. It potentially provides better prediction than Child-Pugh classification and MELD score.

Co-expression of active human cytochrome P450 1A2 and cytochrome P450 reductase on the cell surface of Escherichia coli.

BACKGROUND: Human cytochrome P450 (CYP) enzymes mediate the first step in the breakdown of most drugs and are strongly involved in drug-drug interactions, drug clearance and activation of prodrugs. Their biocatalytic behavior is a key parameter during drug development which requires preparative synthesis of CYP related drug metabolites. However, recombinant expression of CYP enzymes is a challenging bottleneck for drug metabolite biosynthesis. Therefore, we developed a novel approach by displaying human cytochrome P450 1A2 (CYP1A2) and cytochrome P450 reductase (CPR) on the surface of Escherichia coli. RESULTS: To present human CYP1A2 and CPR on the surface, we employed autodisplay. Both enzymes were displayed on the surface which was demonstrated by protease and antibody accessibility tests. CPR activity was first confirmed with the protein substrate cytochrome c. Cells co-expressing CYP1A2 and CPR were capable of catalyzing the conversion of the known CYP1A2 substrates 7-ethoxyresorufin, phenacetin and the artificial substrate luciferin-MultiCYP, which would not have been possible without interaction of both enzymes. Biocatalytic activity was strongly influenced by the composition of the growth medium. Addition of 5-aminolevulinic acid was necessary to obtain a fully active whole cell biocatalyst and was superior to the addition of heme. CONCLUSION: We demonstrated that CYP1A2 and CPR can be co-expressed catalytically active on the cell surface of E. coli. It is a promising step towards pharmaceutical applications such as the synthesis of drug metabolites.