Journal of Pharmaceutical Analysis - 华体会 iOS下载

COVID-19 vaccine candidates and vaccine development platforms available worldwide

2021-09-14

Nilgun Duman, Zahraa ALzaidi, Busra Aynekin, Duygu Taskin, Busra Demirors, Abdulbaki Yildirim, Izem Olcay Sahin, Faik Bilgili, Eda Tahir Turanli, Tommaso Beccari, Matteo Bertelli, Munis Dundar The pandemic caused by the worldwide spread of the coronavirus, which first appeared in 2019, has been named coronavirus disease 19 (COVID-19). More than 4.5 million deaths have been recorded due to the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), according to the World Health Organization. COVID-19 Dashboard in September 2021. Apart from the wildtype, other variations have been successfully transmitted early in the outbreak although they were not discovered until March 2020. Modifications in the SARS-CoV-2 genetic material, such as mutation and recombination, have the ability to modify the viral life span, along with transitivity, cellular tropism, and symptom severity. Several processes are involved in introducing novel vaccines to the population, including vaccine manufacturing, preclinical studies, Food and Drug Administration permission or certification, processing, and marketing. COVID-19 vaccine candidates have been developed by a number of public and private groups employing a variety of strategies, such as RNA, DNA, protein, and viral vectored vaccines. This comprehensive review, which included the most subsequent evidence on unique features of SARS-CoV-2 and the associated morbidity and mortality, was carried out using a systematic search of recent online databases in order to generate useful knowledge about the COVID-19 updated versions and their consequences on the disease symptoms and vaccine development. Journal of Pharmaceutical Analysis. 2021 11(6): 675-682.

Drug repurposing against coronavirus disease 2019 (COVID-19): A review

2021-09-04

Lianxiang Luo, Qin Qiu, Fangfang Huang, Kaifeng Liu, Yongqi Lan, Xiaoling Li, Yuge Huang, Liao Cui, Hui Luo Since December 2019, severe acute respiratory syndrome coronavirus 2 has been found to be the culprit in the coronavirus disease 2019 (COVID-19), causing a global pandemic. Despite the existence of many vaccine programs, the number of confirmed cases and fatalities due to COVID-19 is still increasing. Furthermore, a number of variants have been reported. Because of the absence of approved anti-coronavirus drugs, the treatment and management of COVID-19 has become a global challenge. Under these circumstances, drug repurposing is an effective method to identify candidate drugs with a shorter cycle of clinical trials. Here, we summarize the current status of the application of drug repurposing in COVID-19, including drug repurposing based on virtual computer screening, network pharmacology, and bioactivity, which may be a beneficial COVID-19 treatment. Journal of Pharmaceutical Analysis. 2021 11(6): 683-690.

Possible therapeutic targets and promising drugs based on unsymmetrical hetaryl-substituted porphyrins to combat SARS-CoV-2

2021-08-05

Yury A. Gubarev, Natalya Sh. Lebedeva, Elena S. Yurina, Sergey A. Syrbu, Aleksey N. Kiselev, Mikhail A. Lebedev Coronavirus disease 2019 is a serious disease that causes acute respiratory syndrome and negatively affects the central nervous system. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) crosses the blood-brain barrier due to the spike (S) protein on the surface of the viral particles. Thus, it is important to develop compounds that not only have an inhibitory effect but are also capable of completely deactivating the S protein function. This study describes the purposeful modification of porphyrins and proposes compounds, asymmetrically hetaryl-substituted porphyrins with benzothiazole, benzoxazole, and N-methylbenzimidazole residues, to deactivate the S protein functions. Molecular docking of SARS-CoV-2 proteins with hetaryl-substituted porphyrins showed that the viral S protein, nucleocapsid (N) protein, and non-structural protein 13 (nsp13) exhibited the highest binding affinity.Hetaryl-substituted porphyrins form strong complexes (13–14 kcal/mol) with the receptor-binding domain of the S protein, while the distance from the porphyrins to the receptor-binding motif (RBM) does not exceed 20 Å; therefore, RBM can be oxidized by ¹O₂, which is generated by porphyrin. Hetaryl-substituted porphyrins interact with the N protein in the serine/arginine-rich region, and a number of vulnerable amino acid residues are located in the photooxidation zone. This damage complicates the packaging of viral RNA into new virions. High-energy binding of hetaryl-substituted porphyrins with the N- and C-terminal domains of nsp13 was observed. This binding blocks the action of nsp13 as an enzyme of exoribonuclease and methyltransferase, thereby preventing RNA replication and processing. A procedure for the synthesis of hetaryl-substituted porphyrins was developed, new compounds were obtained, their structures were identified, and their photocatalytic properties were studied. Journal of Pharmaceutical Analysis. 2021 11(6): 691-698.

Overoxidized poly(3,4-ethylenedioxythiophene)–gold nanoparticles–graphene-modified electrode for the simultaneous detection of dopamine and uric acid in the presence of ascorbic acid

Junqiang Pan, Mei Liu, Dandan Li, Haonan Zheng, Dongdong Zhang — 2021-09-17

Junqiang Pan, Mei Liu, Dandan Li, Haonan Zheng, Dongdong Zhang An innovative, ternary nanocomposite composed of overoxidized poly(3,4-ethylenedioxythiophene) (OPEDOT), gold nanoparticles (AuNPs), and electrochemically reduced graphene oxide (ERGO) was prepared on a glassy carbon electrode (GCE) (OPEDOT–AuNPs–ERGO/GCE) through homogeneous chemical reactions and heterogeneous electrochemical methods. The morphology, composition, and structure of this nanocomposite were characterized by transmission electron microscopy, scanning electron microscopy, X-ray diffraction, and X-ray photoelectron spectroscopy. The electrochemical properties of the OPEDOT–AuNPs–ERGO/GCE were investigated by cyclic voltammetry using potassium ferricyanide and hexaammineruthenium(III) chloride redox probe systems. This modified electrode shows excellent electro-catalytic activity for dopamine (DA) and uric acid (UA) under physiological pH conditions, but inhibits the oxidation of ascorbic acid (AA). Linear voltammetric responses were obtained when DA concentrations of approximately 4.0–100 μM and UA concentrations of approximately 20–100 μM were used. The detection limits (S/N=3) for DA and UA were 1.0 and 5.0 μM, respectively, under physiological conditions and in the presence of 1.0 mM of AA. This developed method was applied to the simultaneous detection of DA and UA in human urine, where satisfactory recoveries from 96.7% to 105.0% were observed. This work demonstrates that the developed OPEDOT–AuNPs–ERGO ternary nanocomposite, with its excellent ion-selectivity and electro-catalytic activity, is a promising candidate for the simultaneous detection of DA and UA in the presence of AA in physiological and pathological studies. Journal of Pharmaceutical Analysis. 2021 11(6): 699-708.

Qualitative analysis of chemical components in Lianhua Qingwen capsule by HPLC-Q Exactive-Orbitrap-MS coupled with GC-MS

Shuai Fu, Rongrong Cheng, Zixin Deng, Tiangang Liu — 2021-01-30

Shuai Fu, Rongrong Cheng, Zixin Deng, Tiangang Liu The Lianhua Qingwen (LHQW) capsule is a popular traditional Chinese medicine for the treatment of viral respiratory diseases. In particular, it has been recently prescribed to treat infections caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, due to its complex composition, little attention has been directed toward the analysis of chemical constituents present in the LHQW capsule. This study presents a reliable and comprehensive approach to characterizing the chemical constituents present in LHQW by high-performance liquid chromatography-Q Exactive-Orbitrap mass spectrometry (HPLC-Q Exactive-Orbitrap-MS) coupled with gas chromatography-mass spectrometry (GC-MS). An automated library alignment method with a high mass accuracy (within 5 ppm) was used for the rapid identification of compounds. A total of 104 compounds, consisting of alkaloids, flavonoids, phenols, phenolic acids, phenylpropanoids, quinones, terpenoids, and other phytochemicals, were successfully characterized. In addition, the fragmentation pathways and characteristic fragments of some representative compounds were elucidated. GC-MS analysis was conducted to characterize the volatile compounds present in LHQW. In total, 17 compounds were putatively characterized by comparing the acquired data with that from the NIST library. The major constituent was menthol, and all the other compounds were terpenoids. This is the first comprehensive report on the identification of the major chemical constituents present in the LHQW capsule by HPLC-Q Exactive-Orbitrap-MS, coupled with GC-MS, and the results of this study can be used for the quality control and standardization of LHQW capsules. Journal of Pharmaceutical Analysis. 2021 11(6): 709-716.

Simultaneous determination of 35 constituents and elucidation of effective constituents in a multi-herb Chinese medicine formula Xiaoer-Feire-Kechuan

2021-01-29

Zhanpeng Shang, Lulu Xu, Yi Kuang, Yan Lin, Shuang Liu, Long Sun, Tao Bo, Min Ye, Xue Qiao Xiaoer-Feire-Kechuan (XFK) is an 11-herb Chinese medicine formula to treat cough and pulmonary inflammation. The complicated composition rendered its chemical analysis and effective-component elucidation. In this study, we combined quantitative analysis and bioactivity test to reveal the anti-inflammatory constituents of XFK. First, UPLC-DAD and UHPLC/Q-Orbitrap-MS methods were established and validated to quantify 35 analytes (covering 9 out of 11 herbs) in different XFK formulations. Parallel reaction monitoring mode built in Q-Orbitrap-MS was used to improve the sensitivity and selectivity. Then, anti-inflammatory activities of the 35 analytes were analyzed using in vitro COX-2 inhibition assay. Finally, major analytes forsythosides H, I, A (8–10), and baicalin (15) (total contents varied from 21.79 to 91.20 mg/dose in different formulations) with significant activities (inhibitory rate ≥ 80%) were proposed as the anti-inflammatory constituents of XFK. The present study provided an effective strategy to discover effective constituents of multi-herb formulas. Journal of Pharmaceutical Analysis. 2021 11(6): 717-725.

Detection and quantitation of host cell proteins in monoclonal antibody drug products using automated sample preparation and data-independent acquisition LC-MS/MS

2021-05-20

Lisa Strasser, Giorgio Oliviero, Craig Jakes, Izabela Zaborowska, Patrick Floris, Meire Ribeiro da Silva, Florian Füssl, Sara Carillo, Jonathan Bones Ensuring the removal of host cell proteins (HCPs) during downstream processing of recombinant proteins such as monoclonal antibodies (mAbs) remains a challenge. Since residual HCPs might affect product stability or safety, constant monitoring is required to demonstrate their removal to be below the regulatory accepted level of 100 ng/mg. The current standard analytical approach for this procedure is based on ELISA; however, this approach only measures the overall HCP content. Therefore, the use of orthogonal methods, such as liquid chromatography-mass spectrometry (LC-MS), has been established, as it facilitates the quantitation of total HCPs as well as the identification and quantitation of the individual HCPs present. In the present study, a workflow for HCP detection and quantitation using an automated magnetic bead-based sample preparation, in combination with a data-independent acquisition (DIA) LC-MS analysis, was established. Employing the same instrumental setup commonly used for peptide mapping analysis of mAbs allows for its quick and easy implementation into pre-existing workflows, avoiding the need for dedicated instrumentation or personnel. Thereby, quantitation of HCPs over a broad dynamic range was enabled to allow monitoring of problematic HCPs or to track changes upon altered bioprocessing conditions. Journal of Pharmaceutical Analysis. 2021 11(6): 726-731.

An LC-MS/MS method for simultaneous analysis of the cystic fibrosis therapeutic drugs colistin, ivacaftor and ciprofloxacin

Huiya Yuan, Shihui Yu, Guihong Chai, Junting Liu, Qi (Tony) Zhou — 2021-02-28

Huiya Yuan, Shihui Yu, Guihong Chai, Junting Liu, Qi (Tony) Zhou Inhaled antibiotics such as colistin and ciprofloxacin are increasingly used to treat bacterial lung infections in cystic fibrosis patients. In this study, we established and validated a new HPLC-MS/MS method that could simultaneously detect drug concentrations of ciprofloxacin, colistin and ivacaftor in rat plasma, human epithelial cell lysate, cell culture medium, and drug transport media. An aliquot of 200 μL drug-containing rat plasma or cell culture medium was treated with 600 μL of extraction solution (acetonitrile containing 0.1% formic acid and 0.2% trifluoroacetic acid (TFA)). The addition of 0.2% TFA helped to break the drug-protein bonds. Moreover, the addition of 0.1% formic acid to the transport medium and cell lysate samples could significantly improve the response and reproducibility. After vortexing and centrifuging, the sample components were analyzed by HPLC-MS/MS. The multiple reaction monitoring mode was used to detect the following transitions: 585.5–101.1 (colistin A), 578.5–101.1 (colistin B), 393.2–337.2 (ivacaftor), 332.2–314.2 (ciprofloxacin), 602.3–101.1 (polymyxin B1 as internal standard (IS)) and 595.4–101.1 (polymyxin B2 as IS). The running time of a single sample was only 6 min, making this a time-efficient method. Linear correlations were found for colistin A at 0.029–5.82 μg/mL, colistin B at 0.016–3.14 μg/mL, ivacaftor at 0.05–10.0 μg/mL, and ciprofloxacin at 0.043–8.58 μg/mL. Accuracy, precision, and stability of the method were within the acceptable range. This method would be highly useful for research on cytotoxicity, animal pharmacokinetics, and in vitro drug delivery. Journal of Pharmaceutical Analysis. 2021 11(6): 732-738.

Automatic analytical approach for the determination of 12 illicit drugs and nicotine metabolites in wastewater using on-line SPE-UHPLC-MS/MS

2021-01-22

Jingyuan Wang, Likai Qi, Chenzhi Hou, Tingting Zhang, Mengyi Chen, Haitao Meng, Mengxiang Su, Hui Xu, Zhendong Hua, Youmei Wang, Bin Di In this study, we developed a novel on-line solid phase extraction (SPE)-ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS)-based analytical method for simultaneously quantifying 12 illicit drugs and metabolites (methamphetamine, amphetamine, morphine, codeine, 6-monoacetylmorphine, benzoylecgonine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, cocaine, ketamine, norketamine, and methcathinone) and cotinine (COT) in wastewater samples. The analysis was performed by loading 2 mL of the sample onto an Oasis hydrophilic-lipophilic balance cartridge and using a cleanup step (5% methanol) to eliminate interference with a total run time of 13 min. The isotope-labeled internal standard method was used to quantify the target substances and correct for unavoidable losses and matrix effects during the on-line SPE process. Typical analytical characteristics used for method validation were sensitivity, linearity, precision, repeatability, recovery, and matrix effects. The limit of detection (LOD) and limit of quantification (LOQ) of each target were set at 0.20 ng/L and 0.50 ng/L, respectively. The linearity was between 0.5 ng/L and 250 ng/L, except for that of COT. The intra- and inter-day precisions were <10.45% and 25.64%, respectively, and the relative recovery ranged from 83.74% to 162.26%. The method was used to analyze various wastewater samples from 33 cities in China, and the results were compared with the experimental results of identical samples analyzed using off-line SPE. The difference rate was between 19.91% and −20.44%, and the error range could be considered acceptable. These findings showed that on-line SPE is a suitable alternative to off-line SPE for the analysis of illicit drugs in samples. Journal of Pharmaceutical Analysis. 2021 11(6): 739-745.

Simultaneous enantioseparation and simulation studies of atenolol, metoprolol and propranolol on Chiralpak® IG column using supercritical fluid chromatography

Pranav A. Pandya, Priyanka A. Shah, Pranav S. Shrivastav — 2020-12-21

Pranav A. Pandya, Priyanka A. Shah, Pranav S. Shrivastav Enantioseparation of three β-blockers, i.e., atenolol, metoprolol and propranolol, was studied on amylose tris(3-chloro-5-methylphenylcarbamate) immobilized chiral stationary phase using supercritical fluid chromatography (SFC). The effect of organic modifiers (methanol, isopropanol and their mixture), column temperature and back pressure on chiral separation of β-blockers was evaluated. Optimum chromatographic separation with respect to resolution, retention, and analysis time was achieved using a mixture of CO₂ and 0.1% isopropyl amine in isopropanol: methanol (50:50, V/V), in 75:25 (V/V) ratio. Under the optimized conditions, the resolution factors (R_s) and separation factors (α) were greater than 3.0 and 1.5, respectively. Further, with increase in temperature (25–45 °C) and pressure (100–150 bars) there was corresponding decrease in retention factors (k), α and R_s. However, a reverse trend (α and R_s) was observed for atenolol with increase in temperature. The thermodynamic data from van't Hoff plots revealed that the enantioseparation was enthalpy driven for metoprolol and propranolol while entropy driven for atenolol. To understand the mechanism of chiral recognition and the elution behavior of the enantiomers, molecular docking studies were performed. The binding energies obtained from simulation studies were in good agreement with the elution order found experimentally and also with the free energy values. The method was validated in the concentration range of 0.5–10 μg/mL for all the enantiomers. The limit of detection and limit of quantitation ranged from 0.126 to 0.137 μg/mL and 0.376–0.414 μg/mL, respectively. The method was used successfully to analyze these drugs in pharmaceutical preparations. Journal of Pharmaceutical Analysis. 2021 11(6): 746-756.

Spectroscopic studies of the interaction between phosphorus heterocycles and cytochrome P450

Dumei Ma, Libo Zhang, Yingwu Yin, Yuxing Gao, Qian Wang — 2020-12-21

Dumei Ma, Libo Zhang, Yingwu Yin, Yuxing Gao, Qian Wang P450 fatty acid decarboxylase OleT from Staphylococcus aureus (OleT_SA) is a novel cytochrome P450 enzyme that catalyzes the oxidative decarboxylation of fatty acids to yield primarily terminal alkenes and CO₂ or minor α- and β-hydroxylated fatty acids as side-products. In this work, the interactions between a series of cycloalkyl phosphorus heterocycles (CPHs) and OleT_SA were investigated in detail by fluorescence titration experiment, ultraviolet–visible (UV–vis) and ³¹P NMR spectroscopies. Fluorescence titration experiment results clearly showed that a dynamic quenching occurred when CPH-6, a representative CPHs, interacted with OleT_SA with a binding constant value of 15.2 × 10⁴ M⁻¹ at 293 K. The thermodynamic parameters (ΔH, ΔS and ΔG) showed that the hydrogen bond and van der Waals force played major roles in the interaction between OleT_SA and CPHs. The UV–vis and ³¹P NMR studies indicated the penetration of CPH-6 into the interior environment of OleT_SA, which greatly affects the enzymatic activity of OleT_SA. Therefore, our study revealed an effective way to use phosphorus heterocyclic compounds to modulate the activity of cytochrome P450 enzymes. Journal of Pharmaceutical Analysis. 2021 11(6): 757-763.

Impaired tricarboxylic acid cycle flux and mitochondrial aerobic respiration during isoproterenol induced myocardial ischemia is rescued by bilobalide

Zhe Wang, Fan Zhang, Wei Liu, Ning Sheng, Hua Sun, Jinlan Zhang — 2020-08-26

Zhe Wang, Fan Zhang, Wei Liu, Ning Sheng, Hua Sun, Jinlan Zhang There is an urgent need to elucidate the pathogenesis of myocardial ischemia (MI) and potential drug treatments. Here, the anti-MI mechanism and material basis of Ginkgo biloba L. extract (GBE) were studied from the perspective of energy metabolism flux regulation. Metabolic flux analysis (MFA) was performed to investigate energy metabolism flux disorder and the regulatory nodes of GBE components in isoproterenol (ISO)-induced ischemia-like cardiomyocytes. It showed that [U–¹³C] glucose derived m+2 isotopologues from the upstream tricarboxylic acid (TCA) cycle metabolites were markedly accumulated in ISO-injured cardiomyocytes, but the opposite was seen for the downstream metabolites, while their total cellular concentrations were increased. This indicates a blockage of carbon flow from glycolysis and enhanced anaplerosis from other carbon sources. A Seahorse test was used to screen for GBE components with regulatory effects on mitochondrial aerobic respiratory dysfunction. It showed that bilobalide protected against impaired mitochondrial aerobic respiration. MFA also showed that bilobalide significantly modulated the TCA cycle flux, reduced abnormal metabolite accumulation, and balanced the demand of different carbon sources. Western blotting and PCR analysis showed that bilobalide decreased the enhanced expression of key metabolic enzymes in injured cells. Bilobalide's efficacy was verified by in vivo experiments in rats. This is the first report to show that bilobalide, the active ingredient of GBE, protects against MI by rescuing impaired TCA cycle flux. This provides a new mechanism and potential drug treatment for MI. It also shows the potential of MFA/Seahorse combination as a powerful strategy for pharmacological research on herbal medicine. Journal of Pharmaceutical Analysis. 2021 11(6): 764-775.

Evaluation of the gastrointestinal anti-motility effect of Anacardium occidentale stem bark extract: A mechanistic study of antidiarrheal activity

2020-07-21

Blessing O. Omolaso, Francis S. Oluwole, Olugbenga A. Odukanmi, Julius K. Adesanwo, Ahmed A. Ishola, Kayode E. Adewole Diarrhea is a prevalent gastrointestinal problem associated with fatal implications. It is a huge public health concern that requires better alternatives to current drugs. This study investigated the mechanisms involved in the antidiarrheal activity of Anacardium occidentale (Ao) stem bark extract, a plant commonly used in the management of diarrhea in Nigeria. Methanolic stem bark extract of the plant was partitioned into three fractions: hexane fraction, ethyl acetate fraction (AoEF) and methanol fraction. In vitro studies on the effect of these fractions on guinea pig ileum (GPI) strips, as well as the modulatory effect of AoEF on standard agonists- and antagonists-induced GPI contraction and relaxation, revealed AoEF as the most active fraction. In vivo studies to assess the effect of AoEF on the dopaminergic, muscarinic, and serotonergic pathways were carried out using gastric emptying (GE) and gastrointestinal transit (GT) as experimental end points. AoEF was subjected to GC-MS analysis, while the identified compounds were docked with the muscarinic acetylcholine receptor M3 (CHRM3) using AutodockVina. Results indicated that AoEF inhibited GE and GT via inhibition of CHRM3. In addition, GC-MS analysis revealed the presence of 24 compounds in AoEF, while docking indicated that octadecanoic acid 2-(2-hydroxylethoxy) ethyl ester exhibited the highest binding affinity to CHRM3. This study indicated that the antidiarrheal activity of Ao is through its antimotility effect via the inhibition of the muscarinic pathway. And since none of the identified compounds exhibited higher binding affinity to CHRM3 relative to loperamide, the antimotility activity of these phytoconstituents may be via synergism. Journal of Pharmaceutical Analysis. 2021 11(6): 776-782.

Effect of Shengmai Yin on the DNA methylation status of nasopharyngeal carcinoma cell and its radioresistant strains

2020-12-02

Shiya Liu, Zhiyuan Wang, Daoqi Zhu, Jiabin Yang, Dandan Lou, Ruijiao Gao, Zetai Wang, Aiwu Li, Ying Lv, Qin Fan Shengmai Yin (SMY) is a Chinese herbal decoction that effectively alleviates the side effects of radiotherapy in various cancers and helps achieve radiotherapy's clinical efficacy. In this study, we explored the interaction mechanism among SMY, DNA methylation, and nasopharyngeal carcinoma (NPC). We identified differences in DNA methylation levels in NPC CNE-2 cells and its radioresistant cells (CNE-2R) using the methylated DNA immunoprecipitation array and found that CNE-2R cells showed genome-wide changes in methylation status towards a state of hypomethylation. SMY may restore its original DNA methylation status, and thus, enhance radiosensitivity. Furthermore, we confirmed that the differential gene Tenascin-C (TNC) was overexpressed in CNE-2R cells and that SMY downregulated TNC expression. This downregulation of TNC inhibited NPC cell radiation resistance, migration, and invasion. Furthermore, we found that TNC was hypomethylated in CNE-2R cells and partially restored to a hypermethylated state after SMY intervention. DNA methyltransferases 3a may be the key protein in DNA methylation of TNC. Journal of Pharmaceutical Analysis. 2021 11(6): 783-790.

Novel insights into conjugation of antitumor-active unsymmetrical bisacridine C-2028 with glutathione: Characteristics of non-enzymatic and glutathione S-transferase-mediated reactions

Agnieszka Potęga, Michał Kosno, Zofia Mazerska — 2021-04-05

Agnieszka Potęga, Michał Kosno, Zofia Mazerska Unsymmetrical bisacridines (UAs) are a novel potent class of antitumor-active therapeutics. A significant route of phase II drug metabolism is conjugation with glutathione (GSH), which can be non-enzymatic and/or catalyzed by GSH-dependent enzymes. The aim of this work was to investigate the GSH-mediated metabolic pathway of a representative UA, C-2028. GSH-supplemented incubations of C-2028 with rat, but not with human, liver cytosol led to the formation of a single GSH-related metabolite. Interestingly, it was also revealed with rat liver microsomes. Its formation was NADPH-independent and was not inhibited by co-incubation with the cytochrome P450 (CYP450) inhibitor 1-aminobenzotriazole. Therefore, the direct conjugation pathway occurred without the prior CYP450-catalyzed bioactivation of the substrate. In turn, incubations of C-2028 and GSH with human recombinant glutathione S-transferase (GST) P1-1 or with heat-/ethacrynic acid-inactivated liver cytosolic enzymes resulted in the presence or lack of GSH conjugated form, respectively. These findings proved the necessary participation of GST in the initial activation of the GSH thiol group to enable a nucleophilic attack on the substrate molecule. Another C-2028-GSH S-conjugate was also formed during non-enzymatic reaction. Both GSH S-conjugates were characterized by combined liquid chromatography/tandem mass spectrometry. Mechanisms for their formation were proposed. The ability of C-2028 to GST-mediated and/or direct GSH conjugation is suspected to be clinically important. This may affect the patient's drug clearance due to GST activity, loss of GSH, or the interactions with GSH-conjugated drugs. Moreover, GST-mediated depletion of cellular GSH may increase tumor cell exposure to reactive products of UA metabolic transformations. Journal of Pharmaceutical Analysis. 2021 11(6): 791-798.

Synergistic effects of methyl 2-cyano-3,11-dioxo-18beta-olean-1,-12-dien-30-oate and erlotinib on erlotinib-resistant non-small cell lung cancer cells

2021-06-19

Ebony Nottingham, Elizabeth Mazzio, Sunil Kumar Surapaneni, Shallu Kutlehria, Arindam Mondal, Ramesh Badisa, Stephen Safe, Arun K. Rishi, Mandip Singh Non-small cell lung cancer (NSCLC) is often characterized by an underlying mutation in the epidermal growth factor receptor (EGFR), contributing to aggressive metastatic disease. Methyl 2-cyano-3,11-dioxo-18beta-olean-1,12-dien-30-oate (CDODA-Me), a glycyrrhetinic acid derivative, reportedly improves the therapeutic response to erlotinib (ERL), an EGFR tyrosine kinase inhibitor. In the present study, we performed a series of studies to demonstrate the efficacy of CDODA-Me (2 μM) in sensitizing HCC827R (ERL-resistant) cells to ERL. Herein, we first established the selectivity of ERL-induced drug resistance in the HCC827R cells, which was sensitized when ERL was combined with CDODA-Me (2 μM), shifting the IC₅₀ from 23.48 μM to 5.46 μM. Subsequently, whole transcriptomic microarray expression data demonstrated that the combination of ERL + CDODA-Me elicited 210 downregulated genes (0.44% of the whole transcriptome (WT)) and 174 upregulated genes (0.36% of the WT), of which approximately 80% were unique to the ERL + CDODA-Me group. Synergistic effects centered on losses to cell cycle progression transcripts, a reduction of minichromosome maintenance complex components (MCM2-7), all key components of the Cdc45·MCM2-7GINS (CMG) complex, and replicative helicases; these effects were tantamount to the upregulation of processes associated with the nuclear factor erythroid 2 like 2 translational response to oxidative stress, including sulfiredoxin 1, heme oxygenase 1, and stress-induced growth inhibitor 1. Collectively, these findings indicate that the synergistic therapeutic effects of ERL + CDODA-Me on resistant NSCLC cells are mediated via the inhibition of mitosis and induction of oxidative stress. Journal of Pharmaceutical Analysis. 2021 11(6): 799-807.

A living cell-based fluorescent reporter for high-throughput screening of anti-tumor drugs

2021-04-07

Ningning Tang, Ling Li, Fei Xie, Ying Lu, Zifan Zuo, Hao Shan, Quan Zhang, Lianwen Zhang Suppression of cellular O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) can repress proliferation and migration of various cancer cells, which opens a new avenue for cancer therapy. Based on the regulation of insulin gene transcription, we designed a cell-based fluorescent reporter capable of sensing cellular O-GlcNAcylation in HEK293T cells. The fluorescent reporter mainly consists of a reporter (green fluorescent protein (GFP)), an internal reference (red fluorescent protein), and an operator (neuronal differentiation 1), which serves as a “sweet switch” to control GFP expression in response to cellular O-GlcNAcylation changes. The fluorescent reporter can efficiently sense reduced levels of cellular O-GlcNAcylation in several cell lines. Using the fluorescent reporter, we screened 120 natural products and obtained one compound, sesamin, which could markedly inhibit protein O-GlcNAcylation in HeLa and human colorectal carcinoma-116 cells and repress their migration in vitro. Altogether, the present study demonstrated the development of a novel strategy for anti-tumor drug screening, as well as for conducting gene transcription studies. Journal of Pharmaceutical Analysis. 2021 11(6): 808-814.