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In light of climate change and the ever-increasing population, salt stress has become a critical issue for agriculture and food security. The use of nano-fertilizers in agriculture is a promising application for salt stress management. Therefore, we investigated a hydroponic experiment to evaluate the effect of different nano-fertilizers: macro-nutrient (K2SO4) and micro-nutrient (ZnO and SiO2) on two alfalfa (Medicago sativa L.) genotypes: (Susceptible: Bulldog 505, and tolerant: Mesa-Sirsa) grown with different salt concentrations (6 and10 dS m−1) in split-split design. The results demonstrated that nano-K2SO4 enhanced shoot dry weight, plant height, number of flowers, number of tillers, root length, root fresh weight, and root dry weight under both salt levels. Addition of nano-K2SO4 enhanced plant relative water contents and electrolyte leakage with both genotypes under different salt levels. Nano-SiO2 promoted proline and SOD production with high salinity with values of (0.78 and 1.06 µmol g−1 FW) and 191.15 and 143.46 U. g−1 FW under Bulldog and Mesa-Sirsa, respectively. The application of nano-ZnO promoted plant micro-elements under 6 dS m−1 with both genotypes. The incorporation of nano-fertilizers into hydroponic systems provides a promising strategy, especially in regions with low water quality.

Areas contaminated by heavy metals were identified in the El-Gharbia Governorate (District) of Egypt. Identification used remote sensing and Geographical Information Systems (GIS) as the main research tools. Digital Elevation Models (DEM), Landsat 8 and contour maps were used to map physiographic units. Nine soil profiles were sampled in different physiographic units in the study area. Geochemical analysis of the 33 soil samples was conducted using X-ray fluorescence spectrometry (XRF). Vanadium (V), nickel (Ni), chromium (Cr), copper (Cu) and zinc (Zn) concentrations were measured. V, Ni and Cr concentrations exceeded recommended safety values in all horizons of the soil profiles, while Cu had a variable distribution. Zn concentrations slightly exceeded recommended concentration limits. Concentrations were mapped in each physiographic unit using the inverse distance weighted (IDW) function of Arc-GIS 10.1 software. Pollution levels were closely associated with industry and urban areas.

This research studies and assesses the possibility of employing ultra-high performance fiber reinforced concrete (UHPFRC) in exterior beam-column joints (BCJs). Eight specimens with various concrete material characteristics and steel reinforcing details are cast and examined under repeated loads. Normal concrete with seismic reinforcing details is used as a control specimen. For certain specimens, UHPC, UHPFRC with 1% steel fiber, and UHPFRC with 2% steel fiber are poured into all BCJs, and others are poured into the critical zone only. The consequences of removing stirrups from the joint were studied. All specimens' crack patterns, hysteresis and envelope curves, ductility factor, stiffness degradation?, and energy dissipation are assessed and corresponded to the control sample. The results demonstrate that UHPFRC strengthened the joint, prevented crack development and extension and the shear failure in the joint, and formed the plastic hinge in the beams. UHPFRC outperforms normal concrete with seismic reinforcing details and UHPC without steel fiber in bearing capacity, ductility, stiffness, and energy dissipation. UHPFRC with 1% steel fiber enhanced joint behavior, while UHPFRC with 2% steel fiber was better. Casting the whole sample with UHPFRC achieved very little improvement. The presence of stirrups in the UHPFRC beam-column joint has little effect on its properties. It is more economical to cast UHPFRC in the joint zone only and reduce or eliminate these stirrups in the case of UHPFRC.

This research studies and assesses the possibility of employing UHPFRC in exterior beam-column joints (BCJs). Eight specimens with various concrete material characteristics and steel reinforcing details are cast and examined under repeated loads. Normal concrete with seismic reinforcing details is used as a control specimen. For certain specimens, UHPC, UHPFRC with 1% steel fiber, and UHPFRC with 2% steel fiber are poured into all BCJs, and others are poured into the critical zone only. The consequences of removing stirrups from the joint were studied. All specimens' crack patterns, hysteresis and envelope curves, ductility factor, stiffness degradation‎, and energy dissipation are assessed and corresponded to the control sample. The results demonstrate that UHPFRC strengthened the joint, prevented crack development and extension and the shear failure in the joint, and formed the plastic hinge in the beams. UHPFRC outperforms normal concrete with seismic reinforcing details and UHPC without steel fiber in bearing capacity, ductility, stiffness, and energy dissipation. UHPFRC with 1% steel fiber enhanced joint behavior, while UHPFRC with 2% steel fiber was better. Casting the whole sample with UHPFRC achieved very little improvement. The presence of stirrups in the UHPFRC beam-column joint has little effect on its properties. It is more economical to casting UHPFRC in the joint zone only and reduce or eliminate these stirrups in the case of UHPFRC

Systemic lupus erythematosus (SLE) is a complex autoimmune disease. Cytokine gene polymorphisms play an important role in SLE. Thus, this study aimed to investigate the associations between interleukin 6 (IL-6) and interleukin 10 (IL-10) promoter single-nucleotide polymorphisms (SNPs) and their susceptibility to SLE and the implications for plasma levels. We genotyped IL-6-174G/C (rs1800795) using mutagenically separated polymerase chain reaction (MS-PCR) and IL-10-1082G/A (rs1800896) and -819C/T (rs1800871) using sequence specific primer polymerase chain reaction (SSP-PCR) in 100 Egyptian patients and 119 controls. The plasma levels of IL-6 and IL-10 were measured by enzyme-linked immunosorbent assay (ELISA). There was significant increase in the frequency of IL-6 (-174) GG genotype (P < 0.05) and G allele (P < 0.01) compared to controls. A significant increase in the distribution of IL-10 (-1082G/A) GG (P < 0.05) and AA (P < 0.05) genotypes and a significant reduction in the frequency of GA genotype (P < 0.05) was found in SLE patients. The mean serum concentration of IL-6 (P < 0.001) and IL-10 (P < 0.001) was significantly elevated in SLE patients compared to healthy controls. There was no  significant association of the most common clinical findings and IL-6 and IL-10 gene polymorphisms in SLE patients. In conclusion, our preliminary study indicated that both GG genotype and G allele of IL-6 (-174G/C) could be considered as risk factors for SLE. In addition, the polymorphisms at IL-10 (-1082 G/G and AA) may play a role in SLE susceptibility in Egyptian patients. Larger prospective studies are needed to confirm our findings. [ABSTRACT FROM AUTHOR]

An abstract of the article "Comparison Between Photodynamic Death of Cultured Melanoma and Melanocyte Cells Using a Vital Stain and the Inflicted Biomolecular Damage Using Synchrotron Infrared Microspectroscopy," by A. Mamoon, R. Talaat, M. Martin, K. Bjornstad, and E. Blakely is presented.

Background: Anti-virulence therapy is a promising strategy to treat multi-drug resistant (MDR) pathogens. Pseudomonas aeruginosa is a potent opportunistic pathogen because of an array of virulence factors that are regulated by quorum sensing systems., Methods: The virulence features of four multi-drug resistant P. aeruginosa strains were investigated upon exposure to the sub-lethal dose of gamma rays (1 kGy), and sub-inhibitory concentrations of bioactive metabolites recovered from local halophilic strains in comparison to control. Then, the gene expression of AHL-mediated quorum sensing systems (las/rhl) was quantitatively determined in treated and untreated groups by real-time PCR., Results: The bioactive metabolites recovered from halophilic strains previously isolated from saline ecosystems were identified as Halomonas cupida (Halo-Rt1), H. elongate (Halo-Rt2), Vigibacillus natechei (Halo-Rt3), Sediminibacillus terrae (Halo-Rt4) and H. almeriensis (Halo-Rt5). Results revealed that both gamma irradiation and bioactive metabolites significantly reduced the virulence factors of the tested MDR strains. The bioactive metabolites showed a maximum efficiency for inhibiting biofilm formation and rhamnolipids production whereas the gamma irradiation succeeded in decreasing other virulence factors to lower levels in comparison to control. Quantitative-PCR results showed that AHL-mediated quorum sensing systems (las/rhl) in P. aeruginosa strains were downregulated either by halo-bacterial metabolites or gamma irradiation in all treatments except the upregulation of both lasI internal gene and rhlR intact gene in P. aeruginosa NCR-RT3 and both rhlI internal gene and rhlR intact gene in P. aeruginosa U3 by nearly two folds or more upon exposure to gamma irradiation. The most potent result was observed in the expression of lasI internal gene that was downregulated by more than ninety folds in P. aeruginosa NCR-RT2 after treatment with metabolites of S. terrae (Halo-Rt4). Analyzing metabolites recovered from H. cupida (Halo-Rt1) and H. elongate (Halo-Rt2) using LC-ESI-MS/MS revealed many chemical compounds that have quorum quenching properties including glabrol, 5,8-dimethoxyquinoline-2-carbaldehyde, linoleoyl ethanolamide, agelasine, penigequinolones derivatives, berberine, tetracosanoic acid, and liquidambaric lactone in the former halophile and phloretin, lycoctonine, fucoxanthin, and crassicauline A in the latter one., Conclusion: QS inhibitors can significantly reduce the pathogenicity of MDR P. aeruginosa strains; and thus can be an effective and successful strategy for treating antibiotic resistant traits., (© 2024. The Author(s).)

Quality of life is greatly affected by chronic wounds. It requires more intensive care than acute wounds. Schedule follow-up appointments with their doctor to track healing. Good wound treatment promotes healing and fewer problems. Wound care requires precise and reliable wound measurement to optimize patient treatment and outcomes according to evidence-based best practices. Images are used to objectively assess wound state by quantifying key healing parameters. Nevertheless, the robust segmentation of wound images is complex because of the high diversity of wound types and imaging conditions. This study proposes and evaluates a novel hybrid model developed for wound segmentation in medical images. The model combines advanced deep learning techniques with traditional image processing methods to improve the accuracy and reliability of wound segmentation. The main objective is to overcome the limitations of existing segmentation methods (UNet) by leveraging the combined advantages of both paradigms. In our investigation, we introduced a hybrid model architecture, wherein a ResNet34 is utilized as the encoder, and a UNet is employed as the decoder. The combination of ResNet34's deep representation learning and UNet's efficient feature extraction yields notable benefits. The architectural design successfully integrated high-level and low-level features, enabling the generation of segmentation maps with high precision and accuracy. Following the implementation of our model to the actual data, we were able to determine the following values for the Intersection over Union (IOU), Dice score, and accuracy: 0.973, 0.986, and 0.9736, respectively. According to the achieved results, the proposed method is more precise and accurate than the current state-of-the-art., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Alabdulhafith, Ba Mahel, Samee, Mahmoud, Talaat, Muthanna and Nassef.)

Background: The examination, counting, and classification of white blood cells (WBCs), also known as leukocytes, are essential processes in the diagnosis of many disorders, including leukemia, a kind of blood cancer characterized by the uncontrolled proliferation of carcinogenic leukocytes in the marrow of the bone. Blood smears can be chemically or microscopically studied to better understand hematological diseases and blood disorders. Detecting, identifying, and categorizing the many blood cell types are essential for disease diagnosis and therapy planning. A theoretical and practical issue. However, methods based on deep learning (DL) have greatly helped blood cell classification., Materials and Methods: Images of blood cells in a microscopic smear were collected from GitHub, a public source that uses the MIT license. An end-to-end computer-aided diagnosis (CAD) system for leukocytes has been created and implemented as part of this study. The introduced system comprises image preprocessing and enhancement, image segmentation, feature extraction and selection, and WBC classification. By combining the DenseNet-161 and the cyclical learning rate (CLR), we contribute an approach that speeds up hyperparameter optimization. We also offer the one-cycle technique to rapidly optimize all hyperparameters of DL models to boost training performance., Results: The dataset has been split into two sets: approximately 80% of the data (9,966 images) for the training set and 20% (2,487 images) for the validation set. The validation set has 623, 620, 620, and 624 eosinophil, lymphocyte, monocyte, and neutrophil images, whereas the training set has 2,497, 2,483, 2,487, and 2,499, respectively. The suggested method has 100% accuracy on the training set of images and 99.8% accuracy on the testing set., Conclusion: Using a combination of the recently developed pretrained convolutional neural network (CNN), DenseNet, and the one fit cycle policy, this study describes a technique of training for the classification of WBCs for leukemia detection. The proposed method is more accurate compared to the state of the art., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Houssein, Mohamed, Abdel Samee, Mahmoud, Talaat, Al-Hejri and Al-Tam.)

Introduction: The spread of contradictory health information was a hallmark of the early COVID-19 pandemic. Because of a limited understanding of the disease, its mode of transmission, and its pathogenicity, the public turned to easily accessible and familiar sources of information. Some of these sources included wrong or incomplete information that could increase health risks and incidents of toxicity due to improper information about the usage of disinfectants. The objective of this study was to assess the relationship between sources of information about the COVID-19 pandemic, the related household cleaning and disinfection practices among adult women living in Egypt, and the associated adverse effects of bleach toxicity during a national lockdown., Methods: Through a self-administered online survey, 452 adult women (18 years and older) living in Egypt were recruited from 13 cities between 4 June 2022 and 4 July 2022 to answer the questionnaire. The questionnaire included (41) questions in Arabic and collected data about respondents' household cleaning and disinfection practices to prevent the spread of the SARS-CoV-2 virus and protect their families during the lockdown that started in Egypt in March 2020., Results: The study found that 88.1% (n = 398) of participants reported increased use of disinfectants during the lockdown. Women who chose social media as their primary source of information to learn about disinfection practices reported an increased frequency of respiratory symptoms associated with bleach toxicity (correlation coefficient = 0.10, p-value = 0.03), followed by women who depended on relatives and friends as the primary source of information (correlation coefficient = 0.10, p-value = 0.02)., Conclusion: This study showed that social media is an easily accessible, efficient and fast communication tool that can act as a primary source for individuals seeking medical information compared to other media platforms (e.g., websites, T.V., satellite channels). However, better regulations and monitoring of its content may help limit the harms caused by the misinformation and disinformation spread by these popular platforms, particularly in times of uncertainty and upheaval., (© 2022. The Author(s).)

Hypertension (HTN) is a common health problem and a major cardiovascular risk factor. Accurate measurement of blood pressure (BP) is mandatory for proper diagnosis and follow-up. The aim of this study was to evaluate the accuracy of mercury sphygmomanometer BP-measuring devices in public hospitals in Cairo, Egypt. Fifty public hospitals were included, and 10% of all mercury sphygmomanometer devices in each hospital were tested. Assessment included physical condition (e.g., mercury status, lid of the device, state of the rubber tubes), leakage rate, and calibration accuracy (as compared with a reference device). Devices were approved as accurate when they could successfully pass all three assessment tests. The total number of sphygmomanometer devices was 465. The overall pass rate was 1.3% (six devices). Twenty-five (5.2%) devices passed all of the physical tests, 50 (10.8%) passed the leakage test, and 50 (16.5%) passed the calibration accuracy test. There were 162 (34.8%) devices that showed a high leakage rate (>80 mmHg) and thus were not tested for calibration accuracy. In conclusion, most of the mercury sphygmomanometer devices in hospitals are neglected and not checked regularly for any errors. A plan should be made to gradually replace those failed devices with new, validated, and well-calibrated devices, preferably devices that do not contain mercury., (© 2020. The Author(s).)

Introduction: Rheumatoid arthritis (RA) is a persistent autoimmune disease in which the activity of proinflammatory cytokines and the imbalance, related to the inflammatory process, between elements of bone tissue remodeling such as osteoclasts and osteoblasts play a key role in development of erosions and bone destruction. MicroRNAs are important regulators of skeletal remodeling and are involved in RA pathogenesis. Myomir-206 (miR-206) is unrivalled among the myomirRs, where it is expressed in skeletal muscle and either absent or minimally expressed in other tissues., Material and Methods: This study was designed to analyze the miR-206 expression pattern in peripheral blood mononuclear cells (PBMCs) using quantitative real time polymerase chain reaction and its correlation with IL-16/IL-17 proinflammatory cytokines in two groups - 20 healthy individuals and 30 patients with RA., Results: Elevated expression of miR-206 was observed in RA patients compared with healthy controls ( p < 0.001). A significant increase in both IL-17 and IL-16 serum levels was found in the RA group ( p < 0.01 and p < 0.05; respectively) compared to the control group. miR-206 expression level and IL-17 production were directly positively correlated ( r = 0.491; p < 0.01). ROC analysis of miR-206 showed a cutoff value of 2.7 with 70% sensitivity, 85% specificity, and the area under the curve was 0.802 ( p < 0.001) with the 95% confidence interval from 0. 676 to 0.927., Conclusions: Taken together, our results indicate the importance of miR-206 expression in patients with RA, as a potential new biomarker that affects bone loss/deformity and its collaborative role with proinflammatory cytokines such as IL-16 and IL-17 in RA bone metabolism. Particular interest should be given to further research to determine the contribution of expression of miR-206 in RA pathogenesis., Competing Interests: The authors declare no conflict of interest.

Background: Helicobacter pylori infection has become more and more resistant to conventional first-line treatment regimens. So, there is a considerable interest in evaluating new antibiotic combinations and regimens. Nitazoxanide is an anti-infective drug with demonstrated activity against protozoa and anaerobic bacteria including H. pylori. This work is designed to evaluate the efficacy and safety of a unique triple nitazoxanide-containing regimen as a treatment regimen in Egyptian patients with H. pylori infection., Methods: Two hundred and 24 patients with upper gastrointestinal tract (GIT) dyspeptic symptoms in whom H. pylori -induced GIT disease was confirmed were included in the study. They have been randomized to receive either nitazoxanide 500 mg b.i.d., clarithromycin 500 mg b.i.d., and omeprazole 40 mg twice daily for 14 days or metronidazole 500 mg b.i.d., clarithromycin 500 mg b.i.d., and omeprazole 40  mg twice daily for 14 days. Laboratory evaluation for H. pylori antigen within the stool was performed 6 weeks after cessation of H. pylori treatment regimens to assess the response., Results: The response to treatment was significantly higher in group 1 of nitazoxanide treatment regimen than group 2 of traditional treatment regimen. One hundred and six cases (94.6%) of 112 patients who completed the study in group 1 showed complete cure, while only 63 cases (60.6%) of 104 patients who completed the study in group 2 showed the same response according to per-protocol (PP) analysis (P<.001). The regimen was well tolerated by all the patients enrolled in the study., Conclusion: Nitazoxanide-containing triple therapy is a promising therapy for the first-line eradication of H. pylori. (ClinicalTrials.gov Identifier: NCT02422706)., (© 2017 John Wiley & Sons Ltd.)

Fungi are known to be affected by external environmental stimuli, resulting in different stress response effects, which in turn could be used to enhance its biodegrading ability. In a previous study, ethanol was used to manipulate cell-cell and cell-surface interaction to prevent cell loss and maximize the usage of Penicillium purpurogenum cells in the media, a correlation was drawn between ethanol oxidative stress, surface-bound proteins and fungal adhesion. The present study focuses on a more detailed study of the effect of ethanol on the same fungus. The results show that the presence of Yap1p gene and the detection of an oxidized form of glutathione (GSSG) suggest that a stress response might be involved in the adhesion process. The process of adhesion could be described as a signaling process and it is affected by the germ tube formation as an initial step in adhesion. Protein profile showed polymorphism in surface-bound proteins for cultures amended with ethanol when compared to control cultures. Ethanol also affected the DNA polymorphic profile of DNA, rendering the fungus genetically variable. P. purpurogenum produced phenol oxidase enzyme and could be used to degrade total phenols in olive mill waste water without the formation of biofilm on the surface of the containers.

Thalidomide has anti-inflammatory, immunomodulatory, and anti-angiogenic properties. It has been used to treat a variety of cancers and autoimmune diseases. This study aimed to characterize anti-inflammatory activities of novel thalidomide analogs by exploring their effects on splenocytes proliferation and macrophage functions and their antioxidant activity. MTT assay was used to assess the cytotoxic effect of thalidomide analogs against splenocytes. Tumor necrosis factor (TNF-α) and nuclear factor kappa B (NF-κB-P65) were determined by enzyme-linked immunosorbent assay (ELISA). Nitric oxide (NO) was estimated by colorimetric assay. Antioxidant activity was examined by ORAC assay. Our results demonstrated that thalidomide dithioate analog 2 and thalidomide dithiocarbamate analog 4 produced a slight increase in splenocyte proliferation compared with thalidomide. Thalidomide dithiocarbamate analog 1 is a potent inhibitor of TNF-α production, whereas thalidomide dithiocarbamate analog 5 is a potent inhibitor of both TNF-α and NO. Analog 2 has a pronounced inhibitory effect on NF-κB-P65 production level. All thalidomide analogs showed prooxidant activity against hydroxyl (OH) radical. Analog 1 and thalidomide dithioate analog 3 have prooxidant activity against peroxyl (ROO) radical in relation to thalidomide. On the other hand, analog 4 has a potent scavenging capacity against peroxyl (ROO) radical compared with thalidomide. Taken together, the results of this study suggest that thalidomide analogs might have valuable anti-inflammatory activities with more pronounced effect than thalidomide itself., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Nuclear magnetic resonance (NMR) spectroscopy is playing an increasingly important role in the quantitation of small and large molecules. Recently, we demonstrated that (1)H NMR could be used to quantitate drug metabolites isolated in submilligram quantities from biological sources. It was shown that these metabolites, once quantitated by NMR, were suitable to be used as reference standards in quantitative LC/MS-based assays, hence circumventing the need for radiolabeled material or synthetic standards to obtain plasma exposure estimates in humans and preclinical species. The quantitative capabilities of high-field NMR is further demonstrated in the current study by obtaining the mass balance of fluorinated compounds using (19)F-NMR. Two fluorinated compounds which were radio-labeled with carbon-14 on metabolically stable positions were dosed in rats and urine and feces collected. The mass balance of the compounds was obtained initially by counting the radioactivity present in each sample. Subsequently, the same sets of samples were analyzed by (19)F-NMR, and the concentrations determined by this method were compared with data obtained using radioactivity counting. It was shown that the two methods produced comparable values. To demonstrate the value of this analytical technique in drug discovery, a fluorinated compound was dosed intravenously in dogs and feces and urine collected. Initial profiling of samples showed that this compound was excreted mainly unchanged in feces, and hence, an estimate of mass balance was obtained using (19)F-NMR. The data obtained by this method was confirmed by additional quantitative studies using mass spectrometry. Hence cross-validations of the quantitative (19)F-NMR method by radioactivity counting and mass spectrometric analysis were demonstrated in this study. A strategy outlining the use of fluorinated compounds in conjunction with (19)F-NMR to understand their routes of excretion or mass balance in animals is proposed. These studies demonstrate that quantitative (19)F-NMR could be used as an alternate technique to obtain an estimate of the mass balance of fluorinated compounds, especially in early drug development where attrition of the compounds is high, and cost savings could be realized through the use of such a technique rather than employing radioactive compounds. The potential application of qNMR in conducting early human ADME studies with fluorinated compounds is also discussed., (© 2012 American Chemical Society)

It is important to gain an understanding of the pharmacological activities of metabolite(s) of compounds in development, especially if they are found in systemic circulation in humans. Pharmacological evaluation of metabolites is normally conducted with synthetic standards, which become available during various stages of drug development. However, the synthesis of metabolite standards may be protracted, taking anywhere from several weeks to months to be completed. This often slows down early pharmacological evaluation of metabolites. Once a metabolite(s) is found to possess comparable (or greater) pharmacological activity than the parent compound, additional studies are performed to better understand the implications of circulating pharmacologically active metabolite(s). To conduct some of these studies as early as possible without slowing the progression of a compound in development is important, especially if critical go or no-go decisions impinge on the outcomes from these studies. Early pharmacological evaluation of significant metabolites is hereby proposed to be conducted in the drug discovery stage so that all pertinent studies and information can be gathered in a timely manner for decision-making. It is suggested that these major metabolites be isolated, either from biological or chemical sources, and quantified appropriately. For biologically generated metabolites, NMR is proposed as the tool of choice to quantitate these metabolites before their evaluation in pharmacological assays. For metabolites that have the same UV characteristics as the parent compound, quantitation can be conducted using UV spectroscopy instead of NMR. In this article, we propose a strategy that could be used to determine the pharmacological activities of metabolites isolated in submilligram quantities.

The study was initiated as an observation of incomplete extraction recovery of N-(4-(3-chloro-4-(2-pyridinylmethoxy)anilino)-3-cyano-7-ethoxy-6-quinolyl)-4-(dimethylamino)-2-butenamide (HKI-272) from human plasma. The objective of this study was to 1) identify the binding site(s) of HKI-272 to human plasma protein(s); 2) characterize the nature of the binding; and 3) evaluate the potential reversibility of the covalent binding. After incubation of [(14)C]HKI-272 with human plasma, the mixture was directly injected on liquid chromatography/mass spectrometry (LC/MS), and an intact molecular mass of HKI-272 human serum albumin (HSA) adduct was determined to be 66,999 Da, which is 556 Da (molecular mass of HKI-272) larger than the measured molecular mass of HSA (66,443 Da). For peptide mapping, the incubation mixture was separated with SDS-polyacrylamide gel electrophoresis followed by tryptic digestion combined with LC/tandem MS. A radioactive peptide fragment, LDELRDEGKASSAK [amino acid (AA) residue 182-195 of albumin], was confirmed to covalently bind to HKI-272. In addition, after HCl hydrolysis, a radioactive HKI-272-lysine adduct was identified by LC/MS. After combining the results of tryptic digestion and HCl hydrolysis, the AA residue of Lys190 of HSA was confirmed to covalently bind to HKI-272. A standard HKI-272-lysine was synthesized and characterized by NMR. The data showed that the adduct was formed via Michael addition with the epsilon-amine of lysine attacking to the beta-carbon of the amide moiety of HKI-272. Furthermore, reversibility of the covalent binding of HKI-272 to HSA was shown when a gradual release of HKI-272 was observed from protein pellet of HKI-272-treated human plasma after resuspension in phosphate buffer, pH 7.4, at 37 degrees C for 18 h.

Methylnaltrexone (MNTX) is a peripherally acting mu-opioid receptor antagonist and is currently indicated for the treatment of opioid-induced constipation in patients with advanced illness who are receiving palliative care, when response to laxative therapy has not been sufficient. Sulfation to MNTX-3-sulfate (M2) and carbonyl reduction to methyl-6alpha-naltrexol (M4) and methyl-6beta-naltrexol (M5) are the primary metabolic pathways for MNTX in humans. The objectives of this study were to investigate MNTX in vitro metabolism in human and nonclinical species and to identify the human enzymes involved in MNTX metabolism. Of the five commercially available sulfotransferases investigated, only SULT2A1 and SULT1E1 catalyzed M2 formation. Formation of M4 and M5 was catalyzed by NADPH-dependent hepatic cytosolic enzymes, which were identified using selective chemical inhibitors (10 and 100 microM) for aldo-keto reductase (AKR) isoforms, short-chain dehydrogenase/reductase including carbonyl reductase, alcohol dehydrogenase, and quinone oxidoreductase. The results were then compared with the effects of the same inhibitors on 6beta-naltrexol formation from naltrexone, a structural analog of MNTX, which is catalyzed mainly by AKR1C4. The AKR1C inhibitor phenolphthalein inhibited MNTX and naltrexone reduction up to 98%. 5beta-Cholanic acid 3alpha,7alpha-diol, the AKR1C2 inhibitor, and medroxyprogesterone acetate, an inhibitor of AKR1C1, AKR1C2, and AKR1C4, inhibited MNTX reduction up to 67%. Other inhibitors were less potent. In conclusion, the carbonyl reduction of MNTX to M4 and M5 in hepatic cytosol was consistent with previous in vivo observations. AKR1C4 appeared to play a major role in the carbonyl reduction of MNTX, although multiple enzymes in the AKR1C subfamily may be involved. Human SULT2A1 and SULT1E1 were involved in MNTX sulfation.

Methylnaltrexone (MNTX), a selective mu-opioid receptor antagonist, functions as a peripherally acting receptor antagonist in tissues of the gastrointestinal tract. This report describes the metabolic fate of [(3)H]MNTX or [(14)C]MNTX bromide in mice, rats, dogs, and humans after intravenous administration. Separation and identification of plasma and urinary MNTX metabolites was achieved by high-performance liquid chromatography-radioactivity detection and liquid chromatography/mass spectrometry. The structures of the most abundant human metabolites were confirmed by chemical synthesis and NMR spectroscopic analysis. Analysis of radioactivity in plasma and urine showed that MNTX underwent two major pathways of metabolism in humans: sulfation of the phenolic group to MNTX-3-sulfate (M2) and reduction of the carbonyl group to two epimeric alcohols, methyl-6alpha-naltrexol (M4) and methyl-6beta-naltrexol (M5). Neither naltrexone nor its metabolite 6beta-naltrexol were detected in human plasma after administration of MNTX, confirming an earlier observation that N-demethylation was not a metabolic pathway of MNTX in humans. The urinary metabolite profiles in humans were consistent with plasma profiles. In mice, the circulating and urinary metabolites included M5, MNTX-3-glucuronide (M9), 2-hydroxy-3-O-methyl MNTX (M6), and its glucuronide (M10). M2, M5, M6, and M9 were observed in rats. Dogs produced only one metabolite, M9. In conclusion, MNTX was not extensively metabolized in humans. Conversion to methyl-6-naltrexol isomers (M4 and M5) and M2 were the primary pathways of metabolism in humans. MNTX was metabolized to a higher extent in mice than in rats, dogs, and humans. Glucuronidation was a major metabolic pathway in mice, rats, and dogs, but not in humans. Overall, the data suggested species differences in the metabolism of MNTX.

The recent guidance on "Safety Testing of Drug Metabolites" issued by the U.S. Food and Drug Administration, Center for Drug Evaluation and Research (CDER) has highlighted the importance of identifying and characterizing drug metabolites as early as possible in drug discovery and development. Furthermore, upon identifying significant circulating metabolites in human plasma, it has become important to demonstrate that these metabolites are present at an equal or greater exposure level (area under the curve, AUC) in any one of the preclinical species used in safety testing. Frequently, synthetic standards of metabolites are not available, and hence, obtaining their AUC values can be a challenge. In this report, we demonstrate how combinations of nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography/ultraviolet/mass spectrometry (LC/UV/MS), and plasma pooling methods were used to obtain reliable AUC values of metabolites present in the plasma of preclinical species from short-term safety studies. Plasma pooling methods were compared to the traditional approaches of obtaining quantitative information on the levels of circulating metabolites in preclinical species. The exposure values obtained via sample pooling were comparable to those obtained by traditional methods of analyzing samples individually. In the absence of synthetic chemical standards, calculations of AUC values of metabolites, using either sample pooling or traditional approaches, were achieved through the use of UV detectors. In cases where the UV properties of metabolites were significantly different from their parent compounds, NMR was used as a quantitative tool to obtain exposure values. NMR was found to be useful in quantitating biologically produced metabolites, which could subsequently be used as reference compounds for further quantitative studies. The limitations of UV detectors to obtain exposure estimates are discussed. A practical solution is presented that will enable us to obtain a quantitative assessment of metabolite exposure in humans and coverage in toxicology species, hence, circumventing the use of radiolabeled compounds or authentic chemically synthesized standards of metabolites.

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.

Tigecycline, a novel, first-in-class glycylcycline antibiotic, has been approved for the treatment of complicated intra-abdominal infections and complicated skin and skin structure infections. The pharmacokinetics, metabolism, and excretion of [(14)C]tigecycline were examined in healthy male volunteers. Tigecycline has been shown to bind to bone; thus, to minimize the amount of radioactivity binding to bone and to maximize the recovery of radioactivity, tigecycline was administered intravenously (30-min infusion) as a single 100-mg dose, followed by six 50-mg doses, every 12 h, with the last dose being [(14)C]tigecycline (50 microCi). After the final dose, the pharmacokinetics of tigecycline in serum showed a long half-life (55.8 h) and a large volume of distribution (21.0 l/kg), whereas radioactivity in serum had a shorter half-life (6.9 h) and a smaller volume of distribution (3.3 l/kg). The major route of elimination was feces, containing 59% of the radioactive dose, whereas urine contained 32%. Unchanged tigecycline was the predominant drug-related compound in serum, urine, and feces. The major metabolic pathways identified were glucuronidation of tigecycline and amide hydrolysis followed by N-acetylation to form N-acetyl-9-aminominocycline. The glucuronide metabolites accounted for 5 to 20% of serum radioactivity, and approximately 9% of the dose was excreted as glucuronide conjugates within 48 h. Concentrations of N-acetyl-9-aminominocycline were approximately 6.5% and 11% of the tigecycline concentrations in serum and urine, respectively. Excretion of unchanged tigecycline into feces was the primary route of elimination, and the secondary elimination pathways were renal excretion of unchanged drug and metabolism to glucuronide conjugates and N-acetyl-9-aminominocycline.

A P450 catalyzed N-para-hydroxy metabolite was suggested to be a prerequisite for N-dephenylation occurrence. Although two mechanisms have been proposed to describe this process as a consequence of either a chemical degradation or P450 lead epoxidation of the hydroxy metabolite, direct evidence has not been demonstrated. In this study, we started with a novel technique using a dipeptide, Lys-Phe, to trap the byproduct of N-dephenylation, a quinone-like compound, forming a peptide adduct to facilitate LC/MS characterization. N-dephenylation via chemical degradation was assessed by LC/MS characterization of the resulting (Lys-Phe)(2)-quinone from 4-hydroxyphenyl-2-naphthylamine following interaction with Lys-Phe in pH 7.4 buffer. N-dephenylation mediated by P450 catalysis proposed was investigated in N-para-hydroxy benzodioxane derivative incubated with mouse liver microsomes in the presence of Lys-Phe in 50/50 H(2)(16)O/H(2)(18)O. LC/MS demonstrated that only one of two hydroxy oxygens in the byproduct was exchanged with water and the MS signal intensity of the (16)O labeled peptide adduct was equal to that of (18)O labeled. These observations suggested us that the origin of the oxygen in the byproduct was from water only, not from O(2). Therefore, it appears that N-dephenylation occurs via a stepwise process, namely the substrate is initially metabolized to a N-para-hydroxy metabolite by P450, which was readily oxidized to a quinone imine/iminium chemically or enzymatically, then hydrolyzed resulting in N-dephenylation. However, in our studies, the proposed P450 mechanism involving epoxidation of a N-para-hydroxy metabolite was disproved.

Background: This investigation was undertaken to identify the structure of a novel immunoreactive metabolite derived from fosphenytoin that has been hypothesized previously as present in sera from renally impaired patients receiving this prodrug., Methods: The metabolite was isolated from uremic sera using solid-phase extraction and HPLC. Structural analysis was performed using HPLC-tandem mass spectrometry, nuclear magnetic resonance (NMR), deuterium exchange, and chemical derivatization. Immunoreactivity was evaluated using a fluorescence polarization immunoassay., Results: The metabolite had a parent ion at m/z 457 in the negative-ion mode and fragmented to yield the m/z 251 of phenytoin, as well as other mass fragments of phenytoin. Mass fragments associated with glucuronic acid were also present. The chromatographic peak corresponding to this metabolite demonstrated immunoreactivity sufficient to lead to falsely increased reported values for phenytoin immunoassays. The observed immunoreactivity was also proportional to the relative concentration of the metabolite in collected fractions. Analysis by NMR indicated the presence of phenyl groups with chemical shifts identical to those of phenytoin, as well as the presence of a methylene bridge, which was consistent with the same methylene bridge present on the phosphate ester of fosphenytoin. Comparative analysis of serum samples from renally impaired patients receiving phenytoin vs fosphenytoin using multiple reaction monitoring quantification demonstrated that this metabolite was associated with fosphenytoin administration., Conclusions: A unique immunoreactive oxymethylglucuronide metabolite derived from fosphenytoin has been isolated from sera from uremic patients receiving this prodrug.

The metabolism and distribution of [2,3-(14)C]acrolein were studied in a lactating goat orally administered 0.82 mg/kg of body weight/day for 5 days. Milk, urine, feces, and expired air were collected. The goat was killed 12 h after the last dose, and edible tissues were collected. The nature of the radioactive residues was determined in milk and tissues. All of the identified metabolites were the result of the incorporation of acrolein into the normal, natural products of intermediary metabolism. There was evidence that the three-carbon unit of acrolein was incorporated intact into glucose, and subsequently lactose, and into glycerol. In the case of other natural products, the incorporation of radioactivity appeared to result from the metabolism of acrolein to smaller molecules followed by incorporation of these metabolites into the normal biosynthetic pathways.

The metabolites of [2,3-14C]acrolein in the urine and feces of Sprague-Dawley rats were identified after either intravenous administration in saline at 2.5 mg/kg or oral administration by gavage as an aqueous solution as either single or multiple doses at 2.5 mg/kg or as a single dose of 15 mg/kg. Selected urine and feces samples were pooled by sex and collection interval and profiled by combinations of reverse-phase, anion-exchange, cation-exchange, and ion-exclusion high-performance liquid chromatography (HPLC). Feces were also profiled by size-exclusion chromatography. Metabolites were identified by comparison with well-characterized standards by HPLC and by mass spectrometry. The urinary metabolites were identified as oxalic acid, malonic acid, N-acetyl-S-2-carboxy-2-hydroxyethylcysteine, N-acetyl-S-3-hydroxypropylcysteine, N-acetyl-S-2-carboxyethylcysteine, and 3-hydroxypropionic acid. The fecal radioactivity from the oral dose groups was partitioned into methanol-soluble, water-soluble, and insoluble radioactivity, some of which could be liberated by dilute acid hydrolysis. HPLC analysis of these extracts revealed no discrete metabolites. Size-exclusion chromatography indicated a molecular weight range of 2,000 to 20,000 Da for the radioactivity, which was unaffected by hydrolysis at reflux with 6 M acid or base. This radio-activity was thought to be a homopolymer of acrolein, which was apparently formed in the gastrointestinal tract. The pathways of acrolein metabolism were epoxidation followed by conjugation with glutathione, Michael addition of water followed by oxidative degradation, and glutathione addition to the double bond either following or preceding oxidation or reduction of the aldehyde. The glutathione adducts were further metabolized to the mercapturic acids.